Method for polynucleotide synthesis

ABSTRACT

Methods of forming an internucleotide bond are disclosed. Such methods find use in synthesis of polynucleotides. The method involves contacting a functionalized support with a precursor having an exocyclic amine triaryl methyl protecting group under conditions and for a time sufficient to result in internucleotide bond formation. The functionalized support includes a solid support, a triaryl methyl linker group, and a nucleoside moiety having a reactive site hydroxyl, the nucleoside moiety attached to the solid support via the triaryl methyl linker group. In particular embodiments, the precursor has the structure: 
                         
wherein:
         O and H represent oxygen and hydrogen, respectively   R1 is hydrido, hydroxyl, protected hydroxyl, lower alkyl, modified lower alkyl, or alkoxy,   one of R2 or R3 is a hydroxyl protecting group; and the other of R2 or R3 is a reactive group capable of reacting with the reactive site hydroxyl,   Base is a heterocyclic base having an exocyclic amine group, and   Tram is the exocyclic amine triaryl methyl protecting group.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Agreement No.N39998-01-9-7068. The Government has certain rights in the invention.

RELATED APPLICATIONS

Related subject matter is disclosed in U.S. Patent Applications entitled“Method of Polynucleotide Synthesis Using Modified Support”, (Ser. No.10/652,049); “Cleavable Linker for Polynucleotide Synthesis”, (Ser. No.10/652,063); “Exocyclic Amine Triaryl Methyl Protecting Groups inTwo-Step Polynucleotide Synthesis” (Ser. No. 10/652,064); “PrecursorsFor Two-Step Polynucleotide Synthesis” (Ser. No. 10/652,048); allapplications filed in the names of Dellinger et al. on Aug. 30, 2003,the same day as the instant application, all of which are incorporatedherein by reference in their entireties, provided that, if a conflict indefinition of terms arises, the definitions provided in the presentapplication shall be controlling.

DESCRIPTION

1. Field of the Invention

The invention relates generally to nucleic acid chemistry and to thechemical synthesis of polynucleotide. More particularly, the inventionrelates to providing modified starting materials for use inpolynucleotide synthesis to provide for reduced incidence of undesiredside reactions. The invention is useful in the manufacture of reagentsand devices used in the fields of biochemistry, molecular biology andpharmacology, and in medical diagnostic and screening technologies.

2. Background of the Invention

Solid phase chemical synthesis of DNA fragments is routinely performedusing protected nucleoside phosphoramidites. Beaucage et al. (1981)Tetrahedron Lett. 22:1859. In this approach, the 3′-hydroxyl group of aninitial 5′-protected nucleoside is first covalently attached to thepolymer support. Pless et al. (1975) Nucleic Acids Res. 2:773. Synthesisof the oligonucleotide then proceeds by deprotection of the 5′-hydroxylgroup of the attached nucleoside, followed by coupling of an incomingnucleoside-3′-phosphoramidite to the deprotected hydroxyl group.Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185. The resultingphosphite triester is finally oxidized to a phosphorotriester tocomplete one round of the synthesis cycle. Letsinger et al. (1976) J.Am. Chem. Soc. 98:3655. The steps of deprotection, coupling andoxidation are repeated until an oligonucleotide of the desired lengthand sequence is obtained. This process is illustrated schematically inFIG. 1 (wherein “B” represents a purine or pyrimidine base, “DMT”represents dimethoxytrityl and “iPR” represents isopropyl). Optionally,after the coupling step, the product may be treated with a capping agentdesigned to esterify failure sequences and cleave phosphite reactionproducts on the heterocyclic bases.

The chemical group conventionally used for the protection of nucleoside5′-hydroxyls is dimethoxytrityl, which is removable with acid. Khorana(1968) Pure Appl. Chem. 17:349; Smith et al. (1962) J. Am. Chem. Soc.84:430. This acid-labile protecting group provides a number ofadvantages for working with both nucleosides and oligonucleotides. Forexample, the DMT group can be introduced onto a nucleosideregioselectively and in high yield. Brown et al. (1979) Methods inEnzymol. 68:109. Also, the lipophilicity of the DMT group greatlyincreases the solubility of nucleosides in organic solvents, and thecarbocation resulting from acidic deprotection gives a strongchromophore, which can be used to indirectly monitor couplingefficiency. Matteucci et al. (1980) Tetrahedron Lett. 21:719. Inaddition, the hydrophobicity of the group can be used to aid separationon reverse-phase HPLC. Becker et al. (1985) J. Chromatogr. 326:219.

However, the use of DMT as a hydroxyl-protecting group for conventionaloligonucleotide synthesis has a number of perceived drawbacks. TheN-glycosidic linkages of oligodeoxyribonucleotides are susceptible toacid catalyzed cleavage (Kochetkov et al., Organic Chemistry of NucleicAcids (New York: Plenum Press, 1972)), and even when the protocol isoptimized, recurrent removal of the DMT group with acid duringoligonucleotide synthesis results in depurination. Shaller et al. (1963)J. Am. Chem. Soc. 85:3821. The N-6-benzoyl-protected deoxyadenosinenucleotide is especially susceptible to glycosidic cleavage, resultingin a substantially reduced yield of the final oligonucleotide. Efcavitchet al. (1985) Nucleosides & Nucleotides 4:267. Attempts have been madeto address the problem of acid-catalyzed depurination utilizingalternative mixtures of acids and various solvents; see, for example,Sonveaux (1986) Bioorganic Chem. 14:274. However, this approach has metwith limited success.

McBride et al. (1986) J. Am. Chem. Soc. 108:2040. Also, using theconventional synthesis scheme set forth in FIG. 1 requires additionalsteps per cycle of addition of a nucleotide to the growingpolynucleotide chain, including the post-coupling deprotection step inwhich the DMT group is removed following oxidation of theinternucleotide phosphite triester linkage to a phosphorotriester.

The problems associated with the use of DMT are exacerbated in solidphase oligonucleotide synthesis where “microscale” parallel reactionsare taking place on a very dense, packed surface. Applications in thefield of genomics and high throughput screening have fueled the demandfor precise chemistry in such a context. Side-reactions, which are knownto occur at detectable but acceptable levels during routine synthesis,can rise to unacceptable levels under the conditions required for theseexpanded applications. Thus, increasingly stringent demands are placedon the chemical synthesis cycle as it was originally conceived, and theproblems associated with conventional methods for synthesizingoligonucleotides are rising to unacceptable levels in these expandedapplications.

Recently, alternate schemes for synthesis of polynucleotides have beendescribed. See, e.g. U.S. Pat. No. 6,222,030 to Dellinger et al., U.S.Pat. Appl'n Publ'n No. US2002/0058802 A1 to Dellinger et al., Seio etal. (2001) Tetrahedron Lett. 42 (49):8657-8660. These schemes involveprotecting groups other than DMT at the 3′ or 5′ positions andcorrespondingly different conditions for performing reactions such asdeprotection at the 3′ or 5′ positions. These schemes have theadditional advantage of reducing the number of steps required per cycleof addition of a nucleotide to the growing polynucleotide chain. FIG. 2illustrates such a process having a two-step synthesis cycle,represented in FIG. 2 as a coupling step and a simultaneous deprotectionand oxidation step.

In previously reported methods such as that shown in FIG. 1, the newlysynthesized oligonucleotides containing N-protected nucleobases aretypically deprotected using displacement by nucleophiles such as ammoniaor methylamine. These reagents can have similar properties to (and thusmay not be compatible with) the reagents used for the alternativeremoval of 3′ or 5′ protecting groups in simplified 2-step DNAsynthesis. Also, the anhydrous solvents required for effective couplingreactions may result in lower solubilities of reactive monomers thandesired.

Furthermore, the conditions used in the previously reported two-stepsynthesis (such as shown in FIG. 2) were discovered to result in a lowincidence of other, unexpected (and undesired) side reactions. Removalof the 3′ or 5′ protecting group also resulted in a small amount ofremoval of the N-protecting group from the nucleobase. This prematuredeprotection frees up reactive sites for phosphoramidite coupling andcan result in nucleobase modifications and chain branching.

Thus, what is needed is an improved synthesis of polynucleotides havinga reduced incidence of the undesired side reactions, and providinggreater solubility for the reactive monomers in anhydrous solvents.

SUMMARY OF THE INVENTION

The invention addresses the aforementioned deficiencies in the art, andprovides novel methods for synthesis of polynucleotides. Such methodsinvolve forming an internucleotide bond by contacting a functionalizedsupport having the structure (Ic)

with a precursor having the structure (IIc)Rag—Sugar—Base—Tram  (IIc)

under conditions and for a time sufficient to result in internucleotidebond formation.

In an embodiment, the method of synthesizing polynucleotides furtherincludes, after the internucleotide bond is formed, exposing the resultof the forming an internucleotide bond step to a composition whichconcurrently oxidizes the internucleotide bond and removes a hydroxylprotecting group from the Sugar group.

The functionalized support employed in the current invention typicallyhas the structure (Ic)

Wherein the groups are defined as follows:

is a solid support,

Trl—is a triaryl methyl linker group having one or more substituents onthe aryl groups,

Lnk′—is a linking group linking the solid support and the triaryl methyllinker group, or is a bond linking the solid support and the triarylmethyl linker group,

(Ntd)_(k)—is a polynucleotide moiety having k nucleotide sub-units,wherein k is an integer in the range from zero to about 200,

Lnk—is a linking group linking the triaryl methyl linker group and thepolynucleotide moiety, or is a bond linking the triaryl methyl linkergroup and polynucleotide moiety, and

Nucl—is a nucleoside moiety having a reactive site hydroxyl.

The precursors used in the current invention comprise a heterocyclicbase having an exocyclic amine group and a substituted or unsubstitutedtriaryl methyl protecting group bound to the exocyclic amine group. Theprecursors typically have the structure (IIc):Rag—Sugar—Base—Tram  (IIc)Wherein the groups are defined as follows:

Rag—a reactive group capable of reacting with a reactive site hydroxylof a nucleoside moiety (e.g. on a nascent polynucleotide molecule in theprocess of being synthesized) to result in formation of aninternucleotide bond,

Sugar—a sugar group such as may be found in a nucleotide or nucleotideanalog, typically ribose or 2′-deoxyribose, wherein the sugar group issubstituted with one or more substituents,

Base—a heterocyclic base having an exocyclic amine group, typicallyattached to the sugar group at the 1′ position of the sugar group, and

Tram—a triaryl methyl protecting group, optionally modified with one ormore substituents, the triaryl protecting group bound to theheterocyclic base via the exocyclic amine group.

The present invention provides methods of synthesizing polynucleotidesusing functionalized supports and precursors described herein. Suchmethods involve forming an internucleotide bond by contacting afunctionalized support with a precursor having an exocyclic aminetriaryl protecting group under conditions and for a time sufficient toresult in internucleotide bond formation. In the method, thefunctionalized support comprises a nucleoside moiety having a reactivesite hydroxyl, wherein the nucleoside moiety is attached to a solidsupport via a triaryl methyl linker group.

In an embodiment, the method of synthesizing polynucleotides furtherincludes, after the internucleotide bond is formed, exposing the resultof the forming an internucleotide bond step to a composition whichconcurrently oxidizes the internucleotide bond and removes a hydroxylprotecting group from the Sugar group.

Further information about the groups described above, functionalizedsupports having the structure (Ic), precursors having the structure(IIc), and use of such functionalized supports and precursors insynthesis of polynucleotides is described herein. The use of a triarylmethyl linker group allows the synthesized polynucleotide to be releasedfrom the solid support under acidic conditions. The use of a triarylmethyl protecting group provides for protection of the exocyclic aminesfrom undesirable side reactions during synthesis. The triaryl methylprotecting groups may then be released from the synthesizedpolynucleotide under mildly acidic conditions. In certain embodiments,the release of the polynucleotide from the support and the release ofthe triaryl methyl protecting groups from the synthesized polynucleotideoccur concurrently under acidic conditions.

Additional objects, advantages, and novel features of this inventionshall be set forth in part in the descriptions and examples that followand in part will become apparent to those skilled in the art uponexamination of the following specifications or may be learned by thepractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the materials and methodsparticularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will be understood from thedescription of representative embodiments of the method herein and thedisclosure of illustrative materials for carrying out the method, takentogether with the Figures, wherein

FIG. 1 schematically illustrates prior art synthesis of polynucleotides.

FIG. 2 depicts a synthesis scheme employing a two step synthesis cycle,including a coupling step and a simultaneous deprotection and oxidationstep.

FIG. 3 depicts a method of synthesis of polynucleotides according to thepresent invention.

To facilitate understanding, identical reference numerals have beenused, where practical, to designate corresponding elements that arecommon to the Figures. Figure components are not drawn to scale.

DETAILED DESCRIPTION

Before the invention is described in detail, it is to be understood thatunless otherwise indicated this invention is not limited to particularmaterials, reagents, reaction materials, manufacturing processes, or thelike, as such may vary. It is also to be understood that the terminologyused herein is for purposes of describing particular embodiments only,and is not intended to be limiting. It is also possible in the presentinvention that steps may be executed in different sequence where this islogically possible. However, the sequence described below is preferred.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an insoluble support” includes a plurality of insolublesupports. In this specification and in the claims that follow,

reference will be made to a number of terms that shall be defined tohave the following meanings unless a contrary intention is apparent:

A “nucleotide” refers to a sub-unit of a nucleic acid (whether DNA orRNA or analogue thereof) which includes a phosphate group, a sugar groupand a heterocyclic base, as well as analogs of such sub-units. A“nucleoside” references a nucleic acid subunit including a sugar groupand a heterocyclic base. A “nucleoside moiety” refers to a portion of amolecule having a sugar group and a heterocyclic base (as in anucleoside); the molecule of which the nucleoside moiety is a portionmay be, e.g. a polynucleotide, oligonucleotide, or nucleosidephosphoramidite. A “nucleotide monomer” refers to a molecule which isnot incorporated in a larger oligo- or poly-nucleotide chain and whichcorresponds to a single nucleotide sub-unit; nucleotide monomers mayalso have activating or protecting groups, if such groups are necessaryfor the intended use of the nucleotide monomer. A “polynucleotideintermediate” references a molecule occurring between steps in chemicalsynthesis of a polynucleotide, where the polynucleotide intermediate issubjected to further reactions to get the intended final product, e.g. aphosphite intermediate which is oxidized to a phosphate in a later stepin the synthesis, or a protected polynucleotide which is thendeprotected. An “oligonucleotide” generally refers to a nucleotidemultimer of about 2 to 200 nucleotides in length, while a“polynucleotide” includes a nucleotide multimer having at least twonucleotides and up to several thousand (e.g. 5000, or 10,000)nucleotides in length. It will be appreciated that, as used herein, theterms “nucleoside”, “nucleoside moiety” and “nucleotide” will includethose moieties which contain not only the naturally occurring purine andpyrimidine bases, e.g., adenine (A), thymine (T), cytosine (C), guanine(G), or uracil (U), but also modified purine and pyrimidine bases andother heterocyclic bases which have been modified (these moieties aresometimes referred to herein, collectively, as “purine and pyrimidinebases and analogs thereof”). Such modifications include, e.g.,methylated purines or pyrimidines, acylated purines or pyrimidines, andthe like, or the addition of a protecting group such as acetyl,difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, or the like. Thepurine or pyrimidine base may also be an analog of the foregoing;suitable analogs will be known to those skilled in the art and aredescribed in the pertinent texts and literature. Common analogs include,but are not limited to, 1-methyladenine, 2-methyladenine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N-6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine,2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine,4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine,2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine,8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil,5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine,hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine,6-thiopurine and 2,6-diaminopurine.

The term “alkyl” as used herein, unless otherwise specified, refers to asaturated straight chain, branched or cyclic hydrocarbon group of 1 to24, typically 1-12, carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl,neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl,2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term “lower alkyl” intendsan alkyl group of one to six carbon atoms, and includes, for example,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl,cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term“cycloalkyl” refers to cyclic alkyl groups such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term “modified alkyl” refers to an alkyl group having from one totwenty-four carbon atoms, and further having additional groups, such asone or more linkages selected from ether-, thio-, amino-, phospho-,oxo-, ester-, and amido-, and/or being substituted with one or moreadditional groups including lower alkyl, aryl, alkoxy, thioalkyl,hydroxyl, amino, sulfonyl, thio, mercapto, imino, halo, cyano, nitro,nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl,silyloxy, and boronyl. The term “modified lower alkyl” refers to a grouphaving from one to six carbon atoms and further having additionalgroups, such as one or more linkages selected from ether-, thio-,amino-, phospho-, keto-, ester-, and amido-, and/or being substitutedwith one or more groups including lower alkyl; aryl, alkoxy, thioalkyl,hydroxyl, amino, sulfonyl, thio, mercapto, imino, halo, cyano, nitro,nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl,silyloxy, and boronyl. The term “alkoxy” as used herein refers to asubstituent —O—R wherein R is alkyl as defined above. The term “loweralkoxy” refers to such a group wherein R is lower alkyl. The term“thioalkyl” as used herein refers to a substituent —S—R wherein R isalkyl as defined above.

The term “alkenyl” as used herein, unless otherwise specified, refers toa branched, unbranched or cyclic (e.g. in the case of C₅ and C₆)hydrocarbon group of 2 to 24, typically 2 to 12, carbon atoms containingat least one double bond, such as ethenyl, vinyl, allyl, octenyl,decenyl, and the like. The term “lower alkenyl” intends an alkenyl groupof two to six carbon atoms, and specifically includes vinyl and allyl.The term “cycloalkenyl” refers to cyclic alkenyl groups.

The term “alkynyl” as used herein, unless otherwise specified, refers toa branched or unbranched hydrocarbon group of 2 to 24, typically 2 to12, carbon atoms containing at least one triple bond, such asacetylenyl, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl,t-butynyl, octynyl, decynyl and the like. The term “lower alkynyl”intends an alkynyl group of two to six carbon atoms, and includes, forexample, acetylenyl and propynyl, and the term “cycloalkynyl” refers tocyclic alkynyl groups.

The term “aryl” as used herein refers to an aromatic species containing1 to 5 aromatic rings, either fused or linked, and either unsubstitutedor substituted with 1 or more substituents typically selected from thegroup consisting of lower alkyl, aryl, aralkyl, lower alkoxy, thioalkyl,hydroxyl, thio, mercapto, amino, imino, halo, cyano, nitro, nitroso,azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy,and boronyl; and lower alkyl substituted with one or more groupsselected from lower alkyl, alkoxy, thioalkyl, hydroxyl, thio, mercapto,amino, imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide,sulfone, sulfoxy, phosphoryl, silyl, silyloxy, and boronyl. Typical arylgroups contain 1 to 3 fused aromatic rings, and more typical aryl groupscontain 1 aromatic ring or 2 fused aromatic rings. Aromatic groupsherein may or may not be heterocyclic. The term “aralkyl” intends amoiety containing both alkyl and aryl species, typically containing lessthan about 24 carbon atoms, and more typically less than about 12 carbonatoms in the alkyl segment of the moiety, and typically containing 1 to5 aromatic rings. The term “aralkyl” will usually be used to refer toaryl-substituted alkyl groups. The term “aralkylene” will be used in asimilar manner to refer to moieties containing both alkylene and arylspecies, typically containing less than about 24 carbon atoms in thealkylene portion and 1 to 5 aromatic rings in the aryl portion, andtypically aryl-substituted alkylene. Exemplary aralkyl groups have thestructure —(CH₂)_(j)—Ar wherein j is an integer in the range of 1 to 24,more typically 1 to 6, and Ar is a monocyclic aryl moiety.

The term “electron withdrawing” denotes the tendency of a substituent toattract valence electrons of the molecule of which it is a part, i.e.,an electron-withdrawing substituent is electronegative.

The term “alpha effect,” as in an “alpha effect” nucleophilicdeprotection reagent, is used to refer to an enhancement ofnucleophilicity that is found when the atom adjacent a nucleophilic sitebears a lone pair of electrons. As the term is used herein, anucleophile is said to exhibit an “alpha effect” if it displays apositive deviation from a Bronsted-type nucleophilicity plot. Hoz et al.(1985) Israel J. Chem. 26:313. See also, Aubort et al. (1970) Chem.Comm. 1378; Brown et al. (1979) J. Chem. Soc. Chem. Comm. 171; Buncel etal. (1982) J. Am. Chem. Soc. 104:4896; Edwards et al. (1962) J. Am.Chem. Soc. 84:16; Evanseck et al. (1987) J. Am. Chem Soc. 109:2349. Themagnitude of the alpha effect is dependent upon the electrophile whichis paired with the specific nucleophile. McIsaac, Jr. et al. (1972), J.Org. Chem. 37:1037. Peroxy anions are example of nucleophiles whichexhibit strong alpha effects.

The term “heterocyclic” refers to a five- or six-membered monocyclicstructure or to an eight- to eleven-membered bicyclic structure which iseither saturated or unsaturated. The heterocyclic groups herein may bealiphatic or aromatic. Each heterocyclic group consists of carbon atomsand from one to four heteroatoms selected from the group consisting ofnitrogen, oxygen and sulfur. As used herein, the term “nitrogenheteroatoms” includes any oxidized form of nitrogen, and the quaternizedform of nitrogen. The term “sulfur heteroatoms” includes any oxidizedform of sulfur. Examples of heterocyclic groups include purine,pyrimidine, piperidinyl, morpholinyl and pyrrolidinyl. “Heterocyclicbase” refers to any natural or non-natural heterocyclic moiety that canparticipate in base pairing or base stacking interaction on anoligonucleotide strand.

“Exocyclic” refers to a group situated outside of the ring of a cyclicchemical structure, e.g. a portion of a substituent of the ring isexocyclic to the ring. As used herein, exocyclic amine refers to anamine group that is a substituent of a ring of a heterocyclic base andincludes embodiments in which the nitrogen of the amine group isattached directly to a member of the ring structure and also includesembodiments in which the nitrogen of the amine group may be linked tothe ring structure of the heterocyclic base via an intervening group.

An “internucleotide bond” refers to a chemical linkage between twonucleoside moieties, such as a phosphodiester linkage in nucleic acidsfound in nature, or such as linkages well known from the art ofsynthesis of nucleic acids and nucleic acid analogues. Aninternucleotide bond may comprise a phospho or phosphite group, and mayinclude linkages where one or more oxygen atoms of the phospho orphosphite group are either modified with a substituent or replaced withanother atom, e.g. a sulfur atom or the nitrogen atom of a mono- ordi-alkyl amino group.

“Moiety” and “group” are used to refer to a portion of a molecule,typically having a particular functional or structural feature, e.g. alinking group (a portion of a molecule connecting two other portions ofthe molecule), or an ethyl moiety (a portion of a molecule with astructure closely related to ethane). A “triaryl methyl linker group” asused herein references a triaryl methyl group having one or moresubstituents on the aromatic rings of the triaryl methyl group, whereinthe triaryl methyl group is bonded to two other moieties such that thetwo other moieties are linked via the triaryl methyl group.

“Linkage” as used herein refers to a first moiety bonded to two othermoieties, wherein the two other moieties are linked via the firstmoiety. Typical linkages include ether (—O—), oxo (—C(O)—), amino(—NH—), amido (—N—C(O)—), thio (—S—), phospho (—P—), ester (—O—C(O)—).

“Bound” may be used herein to indicate direct or indirect attachment. Inthe context of chemical structures, “bound” (or “bonded”) may refer tothe existence of a chemical bond directly joining two moieties orindirectly joining two moieties (e.g. via a linking group). The chemicalbond may be a covalent bond, an ionic bond, a coordination complex,hydrogen bonding, van der Waals interactions, or hydrophobic stacking,or may exhibit characteristics of multiple types of chemical bonds. Incertain instances, “bound” includes embodiments where the attachment isdirect and also embodiments where the attachment is indirect.

“Functionalized” references a process whereby a material is modified tohave a specific moiety bound to the material, e.g. a molecule orsubstrate is modified to have the specific moiety; the material (e.g.molecule or support) that has been so modified is referred to as afunctionalized material (e.g. functionalized molecule or functionalizedsupport).

The term “halo” or “halogen” is used in its conventional sense to referto a chloro, bromo, fluoro or iodo substituent.

By “protecting group” as used herein is meant a species which prevents aportion of a molecule from undergoing a specific chemical reaction, butwhich is removable from the molecule following completion of thatreaction. This is in contrast to a “capping group,” which permanentlybinds to a segment of a molecule to prevent any further chemicaltransformation of that segment. As used herein, a “triaryl methylprotecting group” is a substituted or unsubstituted triaryl methyl groupused (or intended to be used) as a protecting group as described ingreater detail elsewhere herein. An “exocyclic amine protecting group”is a protecting group bonded to an exocyclic amine of a heterocyclicbase as provided herein. “Exocyclic amine triaryl methyl protectinggroup” references a triaryl methyl protecting group bonded to anexocyclic amine of a heterocyclic base as provided herein, i.e. asubstituted or unsubstituted triaryl methyl group is bonded to the aminonitrogen of a exocyclic amine group. A “hydroxyl protecting group”refers to a protecting group where the protected group is a hydroxyl.“Reactive site hydroxyl” references a hydroxyl group capable of reactingwith a precursor to result in an internucleotide bond being formed. Intypical embodiments, the reactive site hydroxyl is the terminal5′-hydroxyl during 3′-5′ polynucleotide synthesis and is the 3′-hydroxylduring 5′-3′ polynucleotide synthesis. An “acid labile protectedhydroxyl” is a hydroxyl group protected by a protecting group that canbe removed by acidic conditions. Similarly, an “acid stabile protectedhydroxyl” is a hydroxyl group protected by a protecting group that isnot removed (is stabile) under acidic conditions. A trityl group is atriphenyl methyl group, in which one or more of the phenyl groups of thetriphenyl methyl group is optionally substituted. A “substituted tritylgroup” or a “substituted triphenyl methyl group” is a triphenyl methylgroup on which one of the hydrogens of the phenyl groups of thetriphenyl methyl group is replaced by a substituent.

The term “substituted” as used to describe chemical structures, groups,or moieties, refers to the structure, group, or moiety comprising one ormore substituents. As used herein, in cases in which a first group is“substituted with” a second group, the second group is attached to thefirst group whereby a moiety of the first group (typically a hydrogen)is replaced by the second group.

“Substituent” references a group that replaces another group in achemical structure. Typical substituents include nonhydrogen atoms (halogroups), functional groups (amino, carbonyl, alkoxy, silyl, orsilyloxy), hydrocarbyl groups. Possible substituents are alkyl, loweralkyl, aryl, aralkyl, lower alkoxy, thioalkyl, hydroxyl, halo, cyano,azido, sulfide, sulfone, sulfoxy, silyl, silyloxy, and lower alkyl.

A “group” includes both substituted and unsubstituted forms. Typicalsubstituents include one or more lower alkyl, modified alkyl, anyhalogen, hydroxy, or aryl. Any substituents are typically chosen so asnot to substantially adversely affect reaction yield (for example, notlower it by more than 20% (or 10%, or 5% or 1%) of the yield otherwiseobtained without a particular substituent or substituent combination).

Hyphens, or dashes, are used at various points throughout thisspecification to indicate attachment, e.g. where two named groups areimmediately adjacent a dash in the text, this indicates the two namedgroups are attached to each other. Similarly, a series of named groupswith dashes between each of the named groups in the text indicates thenamed groups are attached to each other in the order shown. Also, asingle named group adjacent a dash in the text indicates the named groupis typically attached to some other, unnamed group. In some embodiments,the attachment indicated by a dash may be, e.g. a covalent bond betweenthe adjacent named groups. In some other embodiments, the dash mayindicate indirect attachment, i.e. with intervening groups between thenamed groups. At various points throughout the specification a group maybe set forth in the text with or without an adjacent dash, (e.g. amidoor amido-, further e.g. Trl or Trl—, yet further e.g. Lnk, Lnk— or—Lnk—) where the context indicates the group is intended to be (or hasthe potential to be) bound to another group; in such cases, the identityof the group is denoted by the group name (whether or not there is anadjacent dash in the text). Note that where context indicates, a singlegroup may be attached to more than one other group (e.g. the Sugargroup, herein; further e.g. where a linkage is intended, such as linkinggroups).

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anon-hydrogen substituent may or may not be present, and, thus, thedescription includes structures wherein a non-hydrogen substituent ispresent and structures wherein a non-hydrogen substituent is notpresent. At various points herein, a moiety may be described as beingpresent zero or more times: this is equivalent to the moiety beingoptional and includes embodiments in which the moiety is present andembodiments in which the moiety is not present. If the optional moietyis not present (is present in the structure zero times), adjacent groupsdescribed as linked by the optional moiety are linked to each otherdirectly. Similarly, a moiety may be described as being either (1) agroup linking two adjacent groups, or (2) a bond linking the twoadjacent groups: this is equivalent to the moiety being optional andincludes embodiments in which the moiety is present and embodiments inwhich the moiety is not present. If the optional moiety is not present(is present in the structure zero times), adjacent groups described aslinked by the optional moiety are linked to each other directly.

Accordingly, an embodiment in accordance with the invention is directedto a method for synthesizing polynucleotides using functionalizedsupports and precursors described herein. The embodiment comprisesforming an internucleotide bond by contacting a functionalized supportwith a precursor having an exocyclic amine triaryl methyl protectinggroup under conditions and for a time sufficient to result in formationof the internucleotide bond. In the method, the functionalized supportcomprises a nucleoside moiety having a reactive site hydroxyl, a triarylmethyl linker group, and a solid support, the nucleoside moiety attachedto the solid support via the triaryl methyl linker group.

In an embodiment, the method of synthesizing polynucleotides furtherincludes, after the internucleotide bond is formed, exposing the resultof the forming an internucleotide bond step to a composition whichconcurrently oxidizes the internucleotide bond and removes a hydroxylprotecting group.

The functionalized support employed in the method of the currentinvention typically has the structure (Ic)

Wherein the groups are defined as follows:

is a solid support,

Trl—is the triaryl methyl linker group, the triaryl methyl linker grouphaving three aryl groups, each of the three aryl groups bound to acentral methyl carbon, at least one of said three aryl groups having oneor more substituents,

Lnk′—is a linking group linking the solid support and the triaryl methyllinker group, or is a bond linking the solid support and the triarylmethyl linker group,

(Ntd)_(k)—is a polynucleotide moiety having k nucleotide sub-units,wherein k is an integer in the range from zero to about 200,

Lnk—is a linking group linking the triaryl methyl linker group and thepolynucleotide moiety, or is a bond directly linking the triaryl methyllinker group and the polynucleotide moiety, and

Nucl—is a nucleoside moiety having a reactive site hydroxyl.

The precursor employed in the method of the current invention comprisesa heterocyclic base having an exocyclic amine group and a substituted orunsubstituted triaryl methyl protecting group bound to the exocyclicamine group. The precursors typically have the structure (IIc)Rag—Sugar—Base—Tram  (IIc)Wherein the groups are defined as follows:

Rag—a reactive group capable of reacting with a reactive site hydroxylof a nucleoside moiety (e.g. on a nascent polynucleotide molecule in theprocess of being synthesized) to result in formation of aninternucleotide bond; the reactive group is typically a phosphorusderivative as described below in reference to structure (IVc),

Sugar—a sugar group such as may be found in a nucleotide or nucleotideanalog, typically ribose, 2′-deoxyribose, arabinose, xylose, or lyxose,wherein the sugar group is substituted with one or more substituents,

Base—a heterocyclic base having an exocyclic amine group, typicallyattached to the sugar group at the 1′ position of the sugar group, and

Tram—a triaryl methyl protecting group, optionally modified with one ormore substituents, the triaryl methyl protecting group bound to theheterocyclic base via the exocyclic amine group.

The sugar group may be any sugar group (or substituted sugar group)known in the art of nucleotide synthesis and nucleotide analogsynthesis. The skilled practitioner will appreciate that polynucleotideanalogs may be accomplished using such known sugars. Representativesugar groups may be selected from monosaccharides, ketoses, aldoses,pentoses (five carbon sugars), hexoses (six carbon sugars), includingany such groups modified by e.g. oxidation, deoxygenation, introductionof other substituents, alkylation and acylation of hydroxyl groups, andchain branching. The sugar group is typically ribose or 2′-deoxyribose,although other sugars may be used. In an embodiment, the sugar isarabinose. In another embodiment, the sugar is selected from xylose orlyxose. In typical embodiments, the sugar group is a monosaccharide;representative monosaccharides include glycerose, dihydroxyacetone,erythrose, erythrulose, xylose, lyxose, arabinose, ribose, xylulose,ribulose, rhamnose, fucose, glucose, mannose, galactose, fructose,sorbose, glucoheptose, galamannoheptose, sedoheptulose, mannoheptulose,and others.

In certain embodiments, the sugar group is a polyhydroxyketone havingthe structure (IIIac)H—[CH(OH)]_(n)—C(═O)—[CH(OH)]_(m)—H  (IIIac)in which n is an integer from 1 to about 5 and m is an integer from 1 toabout 5; provided that one of the hydrogens or hydroxyls in structure(IIIac) is replaced by the reactive group; and provided that theheterocyclic base is directly bound to one of the carbons of structure(IIIac) (thereby replacing a hydrogen or hydroxyl of structure (IIIac)or adding to the carbonyl carbon of structure (IIIac)). It will bereadily apparent to the reader skilled in the art that, in embodimentsin which the heterocyclic base is added to (i.e. bound directly to) thecarbonyl carbon of structure (IIIac), the other groups (e.g. thecarbonyl oxygen) bound to the carbonyl carbon may be changed to preservenormal valency rules for the groups, e.g. to hydroxyl, hydrido, or othersuitable groups. Typically, the polyhydroxyketone has at least threecarbon atoms, typically at least four carbon atoms, more typically atleast five carbon atoms, and typically has up to about eight carbonatoms, more typically up to about ten carbon atoms. In particularembodiments, the sugar group is based on the given structure in thisparagraph but is modified, e.g. by deoxygenation, by introduction ofother substituents (e.g. replacement of a hydrogen or hydroxyl by asubstituent), by alkylation and/or acylation of hydroxyl groups, bychain branching, and by formation of an intramolecular hemiacetal, andby combinations of the above. Also contemplated are sugar groups inwhich the given structure (IIIac) is modified by intramolecularcyclization reaction, e.g. forming a furanose, pyranose, or other ringstructure. As used herein, a sugar group “based on” structure (IIIac)references any structure disclosed in this paragraph, also encompassingthe modifications to structure (IIIac) as described in this paragraph.

In certain embodiments, the sugar group is a polyhydroxyaldehyde havingthe structureH—[CH(OH)]_(n)—C(═O)H  (IIIbc)in which n is an integer from 2 to about 8, typically from 3 to 7, moretypically from 4 to 6; provided that one of the hydrogens or hydroxylsin structure (IIIbc) is replaced by the reactive group; and providedthat the heterocyclic base is directly bound to one of the carbons ofstructure (IIIbc) (thereby replacing a hydrogen or hydroxyl of structure(IIIbc) or adding to the carbonyl carbon of structure (IIIbc)). It willbe readily apparent to the reader skilled in the art that, inembodiments in which the heterocyclic base is added to (i.e. bounddirectly to) the carbonyl carbon of structure (IIIbc), the other groups(e.g. the carbonyl oxygen, the aldehydic hydrogen) bound to the carbonylcarbon may be changed to preserve normal valency rules for the groups,e.g. to hydroxyl, hydrido, or other suitable groups. In particularembodiments, the sugar group is based on the given structure in thisparagraph but is modified, e.g. by deoxygenation, by introduction ofother substituents (e.g. replacement of a hydrogen or hydroxyl by asubstituent), by alkylation and/or acylation of hydroxyl groups, bychain branching, and by formation of an intramolecular hemiacetal, andby combinations of the above. Also contemplated are sugar groups inwhich the given structure (IIIbc) is modified by intramolecularcyclization reaction, e.g. forming a furanose, pyranose, or other ringstructure. As used herein, a sugar group “based on” structure (IIIbc)references any structure disclosed in this paragraph, also encompassingthe modifications to structure (IIIbc) as described in this paragraph.

The sugar group of the precursor is substituted with one or moresubstituents, e.g. the reactive group and the hydroxyl protecting groupas described herein. In typical embodiments, the precursor has thestructure (IVc)

Wherein the groups are defined as follows:

O and H represent oxygen and hydrogen, respectively

R1 is typically hydrido or hydroxyl (or hydroxyl protecting group),wherein when R1 is hydrido, the sugar is 2′-deoxyribose, as will bepresent in DNA synthesis, and when R1 is hydroxyl (or hydroxylprotecting group), the sugar is ribose, as will be present in RNAsynthesis. In certain embodiments, R1 is lower alkyl, modified loweralkyl, or alkoxy.

One of R2 or R3 is a hydroxyl protecting group releasable underconditions of simultaneous deprotection and oxidation during thepolynucleotide synthesis cycle, as further described herein; and theother of R2 or R3 is a reactive group (Rag—) as referenced above withregard to structure (IIc). Under appropriate conditions as describedherein, the reactive group specifically reacts with a reactive sitehydroxyl such that the desired product of the reaction is achieved inacceptable yield. In this regard, “specifically reacts” means that anacceptable amount of precursor reacts as described herein with thenucleoside moiety to result in internucleotide bond formation inacceptable yield. In various embodiments, the acceptable yield is atleast about 5%, at least about 10%, at least about 20%, at least about30%, at least about 50%, or even more, where the percent indicated isthe proportion of precursor incorporated (in moles) over the theoreticalamount of precursor (in moles) that would be incorporated if thereaction was 100% completed, expressed as a percent. In particularembodiments, the reactive group Rag may comprise a leaving group whichis replaced by a portion of the nucleoside moiety as a result of thereaction.

The reactive group typically is a phosphorus derivative capable ofcoupling to a reactive site hydroxyl of a nucleoside moiety (e.g. on anascent polynucleotide molecule in the process of being synthesized). Areactive group that is a phosphorus derivative has the structure (Vc)

Wherein the groups are defined as follows:

The broken line indicates the bond to the sugar group, typically througheither the 3′-O or 5′-O of the sugar group, though other suitable siteson the sugar group may serve to bond to the reactive group, particularlywhen the sugar group is other than a pentose.

X may be a halogen (particularly Cl or Br) or a secondary amino group,NQ1Q2. Preferred phosphorus derivatives are phosphoramidites, where X isNQ 1Q2, and in which Q1 and Q2 may be the same or different and aretypically selected from the group consisting of alkyl, aryl, aralkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, optionallycontaining one or more nonhydrocarbyl linkages such as ether linkages,thio linkages, oxo linkages, amine, azole, and imine linkages, andoptionally substituted on one or more available carbon atoms with anonhydrocarbyl substituent such as cyano, nitro, halo, or the like.Typically, Q1 and Q2 represent lower alkyl, more preferably stericallyhindered lower alkyls such as isopropyl, t-butyl, isobutyl, sec-butyl,neopentyl, tert-pentyl, isopentyl, sec-pentyl, and the like. Moretypically, Q1 and Q2 both represent isopropyl. Alternatively, Q1 and Q2may be linked to form a mono- or polyheterocyclic ring having a total offrom 1 to 3, usually 1 to 2 heteroatoms and from 1 to 3 rings. In such acase, Q1 and Q2 together with the nitrogen atom to which they areattached represent, for example, pyrrolidone, morpholino or piperidino.Usually, Q1 and Q2 have a total of from 2 to 12 carbon atoms. Examplesof specific —NQ1Q2 moieties thus include, but are not limited to,dimethylamine, diethylamine, diisopropylamine, dibutylamine,methylpropylamine, methylhexylamine, methylcyclopropylamine,ethylcyclohexylamine, methylbenzylamine, methylcyclohexylmethylamine,butylcyclohexylamine, morpholine, thiomorpholine, pyrrolidine,piperidine, 2,6-dimethylpiperidine, piperazine, and the like.

Y is typically hydrido or hydrocarbyl (including substitutedhydrocarbyl), typically alkyl, alkenyl, aryl, aralkyl, or cycloalkyl.More typically, Y represents: lower alkyl; benzyl; substituted benzyl;electron-withdrawing β-substituted aliphatic, particularlyelectron-withdrawing β-substituted ethyl such as β-trihalomethyl ethyl,β-cyanoethyl, β-sulfoethyl, β-nitro-substituted ethyl, and the like;electron-withdrawing substituted phenyl, particularly halo-, sulfo-,cyano- or nitro-substituted phenyl; or electron-withdrawing substitutedphenylethyl. Still more typically, Y represents methyl, β-cyanoethyl,methyl-β-cyanoethyl, dimethyl-β-cyanoethyl, phenylsulfonylethyl,methyl-sulfonylethyl, p-nitrophenylsulfonylethyl,2,2,2-trichloro-1,1-dimethylethyl, 2-(4-pyridyl)ethyl,2-(2-pyridyl)ethyl, allyl, 4-methylene-1-acetylphenol,β-thiobenzoylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl,2,2,2-trichloroethyl, p-nitrophenylethyl, p-cyanophenyl-ethyl,9-fluorenylmethyl, 1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl,2-methylthioethyl, 2-(diphenylphosphino)-ethyl, 1-methyl-1-phenylethyl,3-buten-1-yl, 4-(trimethylsilyl)-2-buten-1-yl, cinnamyl,α-methylcinnamyl, and 8-quinolyl.

Still referring to structure (IVc), the hydroxylprotecting group (e.g.on either the 3′-O or 5′-O, that is, designated by one of R2 or R3,respectively) is any suitable protecting group that is known to bereleasable under conditions of simultaneous deprotection and oxidationduring the polynucleotide synthesis cycle. Exemplary protecting groupsthat may be released to free the hydroxyl group during the simultaneousdeprotection and oxidation step are described in U.S. Pat. No. 6,222,030to Dellinger et al.; U.S. Pat. Appl'n Publ'n No. US2002/0058802 A1 toDellinger et al.; and Seio et al. (2001) Tetrahedron Lett. 42(49):8657-8660. In certain embodiments, the protecting groups may becarbonate protecting groups as described in U.S. Pat. No. 6,222,030. Insome embodiments, the protecting groups may be aryl carbonate protectinggroups as described in U.S. Pat. No. 6,222,030. In other embodiments,the protecting groups may be non-carbonate protecting groups asdescribed in U.S. Pat. Appl'n Publ'n No. US2002/0058802 A1, such as forexample, 3′- or 5′-O-silyl or -siloxyl protecting groups, 3′- or5′-O-ester protecting groups, and 3′- or 5′-O-carbamate protectinggroups. The hydroxyl protecting group may be, for example, a protectinggroup which is labile under nucleophilic attack under neutral or mildlybasic conditions. Examples of protecting groups which are labile undernucleophilic attack under neutral or mildly basic conditions are: esterprotecting groups, carbamate protecting groups, siloxane protectinggroups, silane protecting groups, and sulfonate protecting groups thatβ-eliminate. Examples of suitable hydroxyl protecting groups for one ofR2 or R3 in structure (IVc) are described in “Protective Groups inOrganic Synthesis” by T. W. Green, Wiley Interscience.

With regard to the description of R2 and R3 of structure (IVc), it iswell known within the art that synthesis of a polynucleotide maytypically be performed in a 3′ to 5′ direction, or, alternatively, inthe 5′ to 3′ direction. It will be apparent from the description hereingiven ordinary knowledge in the art that the hydroxyl protecting groupand the reactive group designated by R2 and R3 may occupy either the5′-O or 3′-O positions as described above. The synthesis and use of suchalternate embodiments will be readily apparent given the skill in theart and the disclosure herein.

The Base group referenced in both structure (IIc) and in structure (IVc)may be any heterocyclic base that has an exocyclic amine group. Typicalexamples of heterocyclic bases include the naturally occurring purineand pyrimidine bases, e.g., adenine (A), thymine (T), cytosine (C),guanine (G), or uracil (U), as well as modified purine and pyrimidinebases and other heterocyclic bases which have been modified (thesemoieties are sometimes referred to herein, collectively, as “purine andpyrimidine bases and analogs thereof”). Any heterocyclic base having anexocyclic amine which is known in the literature of nucleotide analogsforms the basis for the Base group referenced in structure (IIc) orstructure (IVc). Typical examples include adenine, cytosine, guanine,and common analogs as recited earlier herein, provided the analogretains the exocyclic amine group. Certain nucleotide analogs that arecontemplated in this context include those described in U.S. patentapplication Ser. No. 10/324,409 entitled “Method Of Producing NucleicAcid Molecules With Reduced Secondary Structure”, filed on Dec. 18,2002, and also those described in U.S. patent application Ser. No.09/358,141 entitled “Method Of Producing Nucleic Acid Molecules WithReduced Secondary Structure”, filed on Jul. 20, 1999 now abandoned. Inparticular embodiments, the exocyclic amine group is directly bonded tothe ring structure of the heterocyclic base, that is, the nitrogen ofthe amine group is attached directly to a member of the ring structureof the heterocyclic base. In some embodiments, the amine group may belinked to the ring structure of the heterocyclic base via an interveninggroup, such as an alkyl group or a modified lower alkyl group, e.g. alower alkyl group having additional groups, such as one or more linkagesselected from ether-, thio-, amino-, phospho-, keto-, ester-, andamido-, and/or being substituted with one or more groups including loweralkyl; aryl, alkoxy, thioalkyl, hydroxyl, amino, sulfonyl, nitro, andhalo.

The Base group is typically bound by an N-glycosidic linkage to the 1′carbon of the sugar group, although other configurations are to beencompassed by the invention. In other embodiments, the Base group isbound by a C-glycosidic linkage to the 1′ carbon of the sugar group. Insome embodiments the Base group is bound to a carbon other than the 1′carbon of the sugar group. Other positions of the Base group on thesugar group and other linkages between the Base group and the sugargroup may be practiced by those of skill in the synthesis of nucleotideanalogs given the disclosure herein, especially where analogousstructures having the given heterocyclic base and sugar group are knownin the art.

With reference to structures (IIc) and (IVc), the Tram group is atriaryl methyl protecting group, optionally modified with one or moresubstituents. The triaryl methyl protecting group is bound to theheterocyclic base via the exocyclic amine group. The exocyclic aminegroup has an amino nitrogen which is typically bound to the centralmethyl carbon of the triaryl methyl protecting group. The triaryl methylprotecting group may be substituted or unsubstituted. A substitutedtriaryl methyl group may have one substituent (i.e. a singly substitutedtriaryl methyl group) on one of the aromatic rings of the triaryl methylgroup, or may have multiple substituents (i.e. a multiply substitutedtriaryl methyl group) on one or more of the aromatic rings of thetriaryl methyl group.

The triaryl methyl protecting group is an optionally substituted triarylmethyl protecting group and has the structure (VIc),

wherein the broken line represents a bond to the amino nitrogen of theexocyclic amine group on which the triaryl methyl protecting group is aprotecting group, and R4, R5, and R6 are independently selected fromaromatic ring moieties, each aromatic ring moiety comprising 4-, 5-, or6-membered rings. Each aromatic ring moiety can independently beheterocyclic, non-heterocyclic, polycyclic or part of a fused ringsystem. Each aromatic ring moiety can be unsubstituted or substitutedwith one or more groups each independently selected from the groupconsisting of lower alkyl, aryl, aralkyl, lower alkoxy, thioalkyl,hydroxyl, thio, mercapto, amino, imino, halo, cyano, nitro, nitroso,azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy,and boronyl; and lower alkyl substituted with one or more groupsselected from lower alkyl, alkoxy, thioalkyl, hydroxyl, thio, mercapto,amino, imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide,sulfone, sulfoxy, phosphoryl, silyl, silyloxy, and boronyl. As usedherein, an aromatic ring moiety may be referenced as an “aromatic ringstructure”. As used herein, the “central methyl carbon” of a triarylmethyl group is the carbon bonded directly to the three aromatic ringstructures. Typical triaryl methyl groups that may be employed inembodiments herein are described in U.S. Pat. No. 4,668,777 toCaruthers; use of such groups in accordance with the present inventionis within ordinary skill in the art given the disclosure herein.

In certain embodiments, R4, R5, and R6 are each independently selectedfrom substituted or unsubstituted aromatic groups such as phenyl,biphenyl, naphthanyl, indolyl, pyridinyl, pyrrolyl, thiophenyl, furanyl,annulenyl, quinolinyl, anthracenyl, and the like. In some embodiments,at least one of R4, R5 and R6 is selected from substituted orunsubstituted aromatic groups other than phenyl such as naphthanyl,indolyl, pyridinyl, pyrrolyl, furanyl, annulenyl, quinolinyl,anthracenyl, and the like; in such embodiments zero, one, or two of R4,R5, and R6 are selected from substituted or unsubstituted phenyl.

In some embodiments, R4, R5, and R6 are independently selected fromstructure (VIIc).

In structure (VIIc), the broken line represents the bond to the centralmethyl carbon of the triaryl methyl protecting group, and R7, R8, R9,R10, and R11 are each independently selected from hydrido; lower alkyl,aryl, aralkyl, lower alkoxy, thioalkyl, hydroxyl, thio, mercapto, amino,imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone,sulfoxy, phosphoryl, silyl, silyloxy, and boronyl; and lower alkylsubstituted with one or more groups selected from lower alkyl, alkoxy,thioalkyl, hydroxyl, thio, mercapto, amino, imino, halo, cyano, nitro,nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl,silyloxy, and boronyl.

In particular embodiments, R4, R5, and R6 are each independentlyselected from phenyl, methoxyphenyl, dimethoxyphenyl andtrimethoxyphenyl groups, such that the Tram—group of structure (IIc) orstructure (IVc) may be an unsubstituted trityl group, amonomethoxytrityl group, a dimethoxytrityl group, a trimethoxytritylgroup, a tetramethoxytrityl group, a pentamethoxytrityl group, ahexamethoxytrityl group, and so on.

In particular embodiments, R4, R5, and R6 are each independentlyselected from phenyl, methoxyphenyl groups, dimethoxyphenyl groups,trimethoxyphenyl groups, tetramethoxyphenyl groups, pentamethoxyphenylgroups, or furyanyl groups such that the Tram group of structure (IIc)or structure (IVc) may be an unsubstituted trityl group, amonomethoxytrityl group, a dimethoxytrityl group, a trimethoxyl tritylgroup, a tetramethoxy trityl group, a pentamethoxytrityl group, aanisylphenylfuranylmethyl group, a dianisylfuranylmethyl group, aphenyldifuranylmethyl group, a anisyldifuranylmethyl group or atrifuranylmethyl group.

In certain embodiments, the precursor may be e.g. a portion of anoligonucleotide or polynucleotide, a portion of a di-, tri-, tetra-, orpenta-nucleotide (a small polynucleotide having 2, 3, 4, or 5 nucleotidemonomer subunits). In such embodiments, the precursor typically has thestructure (IIc) described above, provided that the Sugar group isattached to a nucleotide moiety, an oligonucleotide moiety, or apolynucleotide moiety, in which the attached nucleotide moiety,oligonucleotide moiety, or polynucleotide moiety may have appropriateprotecting groups. For example, the precursor may be a di- ortri-nucleotide in which the 3′ or 5′ hydroxyl bears a hydroxylprotecting group that is removed during the deprotection/oxidation step.

Certain embodiments in accordance with the present invention providenovel methods for synthesis of polynucleotides. Such methods involveforming an internucleotide bond by contacting a functionalized supporthaving the structure (Ic)

with a precursor having the structure (IIc)Rag—Sugar—Base—Tram  (IIc)

under conditions and for a time sufficient to result in internucleotidebond formation. In particular embodiments, the reactive group reactswith the available reactive site hydroxyl of the nucleoside moiety toresult in the internucleotide bond being formed between the nucleosidemoiety of the functionalized support and the sugar group of theprecursor.

In an embodiment, the method of synthesizing polynucleotides furtherincludes, after the internucleotide bond is formed, exposing the resultof the forming an internucleotide bond step to a composition whichconcurrently oxidizes the internucleotide bond and removes a hydroxylprotecting group from the Sugar group.

The functionalized support comprises a nucleoside moiety having areactive site hydroxyl, a triaryl methyl linker group, and a solidsupport, the nucleoside moiety attached to the solid support via thetriaryl methyl linker group.

The functionalized support employed in the current invention typicallyhas the structure (Ic)

Wherein the groups are defined as follows:

is a solid support,

Trl—is the triaryl methyl linker group, the triaryl methyl linker grouphaving three aryl groups, each of the three aryl groups bound to acentral methyl carbon, at least one of said three aryl groups having oneor more substituents,

Lnk′—is a linking group linking the solid support and the triaryl methyllinker group, or is a bond linking the solid support and the triarylmethyl linker group,

(Ntd)_(k)—is a polynucleotide moiety having k nucleotide sub-units,wherein k is an integer in the range from zero to about 200,

Lnk—is a linking group linking the triaryl methyl linker group and thepolynucleotide moiety, or is a bond linking the triaryl methyl linkergroup and polynucleotide moiety, and

Nucl—is a nucleoside moiety having a reactive site hydroxyl.

The solid support may comprise any suitable material adapted for itsintended use in polynucleotide synthesis. The solid support should beessentially inert to the conditions of reactions used for thepolynucleotide synthesis. Typically the solid support includes a solidsubstrate having a surface to which the triaryl methyl linker group isbound, directly or indirectly (i.e. via an intermediate moiety ormoieties, e.g. moieties typically referred to in the art variously aslinking groups (e.g. the Lnk′—group), tethers, or spacers; further e.g.a modification layer on the surface), to the surface. The triaryl methyllinker group is attached to the solid support via a bond to one of thearomatic rings of the triaryl methyl linker group, that is, the solidsupport (and any intermediate moieties, e.g. the Lnk′—group) may beconsidered a substituent of one of the aromatic rings. As indicated instructure (Ic), the nucleoside moiety is attached to the solid substratevia the triaryl methyl linker group such that the nucleoside moiety isaccessible to the precursor when the functionalized support is contactedwith a solution containing the precursor.

In certain embodiments, the solid support comprises a solid substrateand a modification layer disposed on (or bound to, directly orindirectly) the substrate, and the triaryl methyl linker group is boundto (directly or indirectly) the modification layer. Such modificationlayer may be formed on the substrate by methods known in the art ofmodifying surface properties of supports used in polynucleotidesynthesis, or known in the art of modifying supports to provide desiredsurface properties. In certain embodiments, the modification layer maybe, e.g. a coating, a material deposited by deposition techniques knownin the art, a hydrophobic layer, or a hydrophilic layer. In particularembodiments, the modification layer comprises a silane group to whichthe triaryl methyl linker group is bound, directly or indirectly, e.g.via any linking group effective to link the triaryl methyl linker groupto the silane group and stable to the conditions used in polynucleotidesynthesis. Particularly contemplated are supports taught in U.S. Pat.No. 6,258,454 to Lefkowitz et al. (2001) as supports having a moietybound to a substrate via a linking group attached to a silane groupbound to the surface of a substrate.

Functionalized supports in accordance with the present invention may bemade using silane modified substrates such as are employed in theLefkowitz '454 patent and modifications thereof. A functional groupattached (directly or indirectly, e.g. via a linking group) to thesilane group on the substrate provides a site for further attachment tothe substrate to occur. The substrate bearing the functional group isthen contacted with a composition having a surface-binding groupattached to a triaryl methyl linker group. The surface-binding group iscapable of reacting with the functional group attached to the substrateto result in attachment of the triaryl methyl linker group to the solidsupport. Of course, other moieties, such as a nucleoside moiety,attached to the triaryl methyl linker group will thusly also be attachedto the solid support. The resulting functionalized support may then beused in polynucleotide synthesis. The functional group attached to thesubstrate will typically be selected from amine, hydroxyl, sulfhydryl,carboxyl, carbonyl, phosphate and thiophosphate, and combinationsthereof. The surface-binding group comprises a group that is chemicallyreactive with (and forms a covalent bond with) the functional groupattached to the substrate. The surface-binding group will typically beselected from succinimidyl ester, isothiocyanate, isocyanate,haloacetamide, dichlorotriazine, maleimide, sulphonyl halide,alkylimidoester, arylimidoester, substituted hydrazine, substitutedhydroxylamine, carbodiimide, acyl halide, anhydride, phosphoramidite,acrylate and acrylamide. Selection of an appropriate surface-bindinggroup will be based on the identity of the functional group attached tothe substrate, and vice versa. Such selection is within the skill ofthose in the art given the disclosure herein.

The solid substrate typically comprises a material that is stable toconditions used in the synthesis of polynucleotides; such materialsinclude, but are not limited to, supports that are typically used forsolid-phase chemical synthesis, e.g. cross-linked polymeric materials(e.g. divinylbenzene styrene-based polymers), agarose (e.g. SEPHAROSEmedia), dextran (e.g. SEPHADEX media), cellulosic polymers,polyacrylamides, silica, glass (such as controlled pore glass “CPG”),ceramics, and the like.

Referring to structure (Ic), the Trl—group is a substituted triarylmethyl linker group and has the structure (XVIc),

wherein the broken line represents a bond via which the rest of thestructure (XVIc) is connected to the nucleoside moiety (e.g. via thepolynucleotide moiety), and R12, R13, and R14 are independently selectedfrom aromatic ring moieties, each aromatic ring moiety comprising 4-,5-, or 6-membered rings, provided that one of R12, R13, and R14 issubstituted by being bonded to the solid support. Each aromatic ringmoiety can independently be heterocyclic, non-heterocyclic, polycyclicor part of a fused ring system. Each aromatic ring moiety can beunsubstituted or substituted with one or more groups each independentlyselected from the group consisting of lower alkyl, aryl, aralkyl, loweralkoxy, thioalkyl, hydroxyl, thio, mercapto, amino, imino, halo, cyano,nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl,silyl, silyloxy, and boronyl; and lower alkyl substituted with one ormore groups selected from lower alkyl, alkoxy, thioalkyl, hydroxyl,thio, mercapto, amino, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, andboronyl; provided that, as noted above, one of R12, R13, and R14 issubstituted by being bound to the solid support, e.g. via a covalentbond between the ring and a surface modification layer of the solidsupport, or further e.g. via a covalent bond between a linking groupattached to the ring and a functional group of the solid support.Typical triaryl methyl groups that may be employed in embodiments hereinare described in U.S. Pat. No. 4,668,777 to Caruthers, again providedthat, as noted above, one of R12, R13, and R14 is substituted by beingbound to the solid support; use of such groups in accordance with thepresent invention is within ordinary skill in the art given thedisclosure herein.

In certain embodiments, R12 and R13 are each independently selected fromsubstituted or unsubstituted aromatic groups such as phenyl, biphenyl,naphthanyl, indolyl, pyridinyl, pyrrolyl, thiophenyl, furanyl,annulenyl, quinolinyl, anthracenyl, and the like, and R14 is selectedfrom substituted aromatic groups such as phenyl, biphenyl, naphthanyl,indolyl, pyridinyl, pyrrolyl, thiophenyl, furanyl, annulenyl,quinolinyl, anthracenyl, and the like. In some embodiments, at least oneof R12, R13 and R14 is selected from substituted or unsubstitutedaromatic groups other than phenyl such as naphthanyl, indolyl,pyridinyl, pyrrolyl, furanyl, annulenyl, quinolinyl, anthracenyl, andthe like; in such embodiments zero, one, or two of R12, R13, and R14 areselected from substituted or unsubstituted phenyl, provided that, asnoted above, one of R12, R13, and R14 is substituted by being bound tothe solid support.

In some embodiments, R12, R13, and R14 are independently selected fromstructure (XVIIc).

In structure (XVIIc), the broken line represents the bond to the centralmethyl carbon of the triaryl methyl linker group, and R15, R16, R17,R18, and R19 are each independently selected from hydrido, lower alkyl,aryl, aralkyl, lower alkoxy, thioalkyl, hydroxyl, thio, mercapto, amino,imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone,sulfoxy, phosphoryl, silyl, silyloxy, and boronyl; and lower alkylsubstituted with one or more groups selected from lower alkyl, alkoxy,thioalkyl, hydroxyl, thio, mercapto, amino, imino, halo, cyano, nitro,nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl,silyloxy, and boronyl, provided that, for R14, one of the groups R15,R16, R17, R18, and R19 denotes the linkage to the solid support, or isthe group via which the triaryl methyl linker group is attached to thesolid support (e.g. through Lnk′).

In particular embodiments, R12, R13, and R14 are each independentlyselected from phenyl, methoxyphenyl, dimethoxyphenyl andtrimethoxyphenyl groups, such that the Trl—group of structure (Ic) maybe a trityl group, a monomethoxytrityl group, a dimethoxytrityl group, atrimethoxytrityl group, a tetramethoxytrityl group, a pentamethoxytritylgroup, a hexamethoxytrityl group and so on; again provided as describedabove that one of R12, R13, and R14 is substituted by being bound to thesolid support.

In particular embodiments, R12, R13, and R14 are each independentlyselected from phenyl, methoxyphenyl groups, dimethoxyphenyl groups,trimethoxyphenyl groups, tetramethoxyphenyl groups, pentamethoxyphenylgroups, or furyanyl groups such that the Trl—group of structure (Ic) maybe a substituted trityl group, a monomethoxytrityl group, adimethoxytrityl group, a trimethoxyl trityl group, a tetramethoxy tritylgroup, a pentamethoxytrityl group, an anisylphenylfuranylmethyl group, adianisylfuranylmethyl group, a phenyldifuranylmethyl group, ananisyldifuranylmethyl group or a trifuranylmethyl group, again providedas described above that one of R12, R13, and R14 is substituted by beingbound to the solid support.

Referring to structure (Ic), (Ntd)_(k)—is a polynucleotide moiety havingk nucleotide sub-units, wherein k is an integer typically in the rangefrom zero to about 200, more typically in the range from about 1 toabout 200, still more typically in the range from about 2 to about 200,still more typically in the range from about 5 to about 200. It will beapparent that during the initial steps of polynucleotide synthesis, kmay be zero or one, and that as the synthesis cycle is repeated, k willget larger. In certain embodiments, k may be up to 60, more typically upto 100, still more typically up to 200, or even more. The polynucleotidemoiety typically may include naturally occurring and/or non-naturallyoccurring heterocyclic bases and may include heterocyclic bases whichhave been modified, e.g. by inclusion of protecting groups or any othermodifications described herein, or the like. As used herein,“polynucleotide moiety” references a series of connected nucleotidesubunits that is a portion of a larger molecule. The polynucleotidemoiety is typically bound to the linking group via a 3′-O— or a 5′-O— ofthe polynucleotide moiety, although other sites of the polynucleotidemoiety are contemplated and are within the scope of the invention. Thepolynucleotide moiety is typically bound to the Nucl—group via a 3′-O—or a 5′-O— of the polynucleotide moiety, although other sites of thepolynucleotide moiety are contemplated and are within the scope of theinvention. Use of such other sites of the polynucleotide moiety by whichthe polynucleotide moiety may be bound to the linking group or theNucl—group are within the skill in the art given the disclosure herein.

Still referring to structure (Ic), the Lnk—group is selected from (1) alinking group linking the central methyl carbon of the triaryl methyllinker group to the polynucleotide moiety (typically at the 5′ or 3′terminal hydroxyl, or other suitable site of the polynucleotide moiety),or (2) a covalent bond between the central methyl carbon of the triarylmethyl linker group and the polynucleotide moiety (e.g. at the 5′ or 3′terminal hydroxyl, or other suitable site of the polynucleotide moiety).In particular embodiments, the Lnk—group may be any appropriate linkinggroup that links the triaryl methyl linker group to the polynucleotidemoiety, the linking group typically selected from (1) a lower alkylgroup; (2) a modified lower alkyl group in which one or more linkagesselected from ether-, oxo-, thio-, amino-, and phospho- is present; (3)a modified lower alkyl substituted with one or more groups includinglower alkyl; aryl, aralkyl, alkoxyl, thioalkyl, hydroxyl, amino,sulfonyl, halo; or (4) a modified lower alkyl substituted with one ormore groups including lower alkyl; alkoxyl, thioalkyl, hydroxyl, amino,sulfonyl, halo, and in which one or more linkages selected from ether-,oxo-, thio-, amino-, and phospho- is present. In certain embodiments,the linking group is a single non-carbon atom, e.g. —O—, or a singlenon-carbon atom with one or more hydrogens attached, e.g. —N(H)—.

Still referring to structure (Ic), the Lnk′—group is selected from (1) alinking group linking the solid support and the triaryl methyl linkergroup (typically Lnk′—is bound to a ring atom of one of the aryl groupsof the triaryl methyl linker group, i.e. the Lnk′—group may beconsidered a substituent of one of the aryl groups of the triaryl methyllinker group); or (2) a covalent bond between the solid support and thetriaryl methyl linker group (e.g. the solid support is bound to a ringatom of one of the aryl groups of the triaryl methyl linker group, i.e.the solid support may be considered a substituent of one of the arylgroups of the triaryl methyl linker group). In particular embodiments,the Lnk′—group may be any appropriate linking group that links thetriaryl methyl linker group to the solid support, the linking grouptypically selected from (1) a lower alkyl group; (2) a modified loweralkyl group in which one or more linkages selected from ether-, oxo-,thio-, amino-, and phospho- is present; (3) a modified lower alkylsubstituted with one or more groups including lower alkyl; aryl,aralkyl, alkoxyl, thioalkyl, hydroxyl, amino, sulfonyl, halo; or (4) amodified lower alkyl substituted with one or more groups including loweralkyl; alkoxyl, thioalkyl, hydroxyl, amino, sulfonyl, halo, and in whichone or more linkages selected from ether-, oxo-, thio-, amino-, andphospho- is present. In certain embodiments, the Lnk′—group is a singlenon-carbon atom, e.g. —O—, or a single non-carbon atom with one or morehydrogens attached, e.g. —N(H)—.

Again referring to structure (Ic), the Nucl—group is a nucleoside moietyhaving a reactive site hydroxyl. The nucleoside moiety is attached tothe solid support via a triaryl methyl linker group. The nucleosidemoiety has a sugar group (the sugar group comprising the reactive sitehydroxyl) and a heterocyclic base. The sugar group of the nucleosidemoiety may be any sugar group described above with reference to theprecursor of structure (IIc), provided that, instead of being bonded tothe reactive group (of structure (IIc)), the sugar group is bound to thesolid support (via the triaryl methyl linker group, and also via theintervening Lnk— and (Ntd)_(k)—groups in the embodiments depicted bystructure (Ic)); further provided that the reactive site hydroxyl is notprotected by a protecting group (although other sites on the sugar groupmay optionally bear protecting groups). In use, the reactive sitehydroxyl typically originates from the immediately preceding iterationof the synthesis cycle, in which the deprotection/oxidation step resultsin the deprotection of a hydroxyl that is then available to be thereactive site hydroxyl in the next iteration of the synthesis cycle. Theheterocyclic base of the nucleoside moiety may be selected from thenaturally occurring purine and pyrimidine bases, e.g., adenine (A),thymine (T), cytosine (C), guanine (G), or uracil (U), or modifiedpurine and pyrimidine bases and other heterocyclic bases which have beenmodified. The heterocyclic base of the nucleoside moiety may include aprotecting group, or may lack any protecting group. The heterocyclicbase is typically bound by an N-glycosidic linkage to the 1′ carbon ofthe sugar group, although other configurations are to be encompassed bythe invention. In other embodiments, the heterocyclic base is bound by aC-glycosidic linkage to the 1′ carbon of the sugar group. In someembodiments the heterocyclic base is bound to a carbon other than the 1′carbon of the sugar group. Other positions of the heterocyclic base (theatom of the heterocyclic base ring via which the heterocyclic base islinked to the sugar group) and other linkages between the heterocyclicbase and the sugar group may be practiced by those of ordinary skill inthe synthesis of nucleotide analogs given the disclosure herein,especially where analogous structures having the given heterocyclic baseand sugar group are known in the art. The Nucl—group is typically boundto the polynucleotide moiety via a 3′-O— or a 5′-O— of the Nucl—group,although other sites of the Nucl—group are contemplated and are withinthe scope of the invention. Use of such other sites of the Nucl—group bywhich the Nucl—group may be bound to the polynucleotide moiety arewithin the skill in the art given the disclosure herein.

In accordance with the invention, a functionalized support suitable foruse in polynucleotide synthesis is provided, wherein the functionalizedsupport comprises a nucleoside moiety bound to a surface of the solidsupport via a triaryl methyl linker group. The functionalized supportmay then be used to perform polynucleotide synthesis. In a method forsynthesis of a polynucleotide, the functionalized support is contactedwith a precursor having the structure (IIc) under conditions and for atime sufficient to allow the precursor to react with an availablereactive site hydroxyl group of the nucleoside moiety. Such reactions inaccordance with the invention described herein may be repeated aplurality of times to result in the synthesized polynucleotide bound tothe surface via a triaryl methyl linker group.

In use, a precursor that has the structure (IIc) is contacted with areactive site hydroxyl of a nucleoside moiety to result in formation ofan internucleotide bond. Such a reaction is generally shown in FIG. 3,as discussed further, below. FIG. 3 schematically illustrates 3′-to-5′synthesis of a polynucleotide using the method of the present invention.In FIG. 3, the

symbol represents the functionalized support as described herein withrespect to structure (Ic) except the Nucl group, which is explicitlydrawn in the figure. That is, the

symbol represents

Lnk—(Ntd)_(k)—. In the figure, the moiety R^(hpg) represents ahydroxylprotecting group releasable under conditions of simultaneousdeprotection and oxidation during the polynucleotide synthesis cycle, asfurther described herein. In FIG. 3, B^(eatpg) represents a heterocyclicbase having an exocyclic amine group, and further having an exocyclicamine triaryl methyl protecting group bonded to the exocyclic aminegroup. As may be seen, in the second step of the synthesis cycle,deprotection and oxidation occur concurrently. The synthesis may becontrasted with that schematically illustrated in FIG. 1, the prior,conventional method, where the conventional synthesis scheme entailsseparate oxidation and deprotection steps. The synthesis also may becontrasted with that schematically illustrated in FIG. 2, showing asynthesis with the previously taught starting materials. Such asynthesis scheme is known in the art, such as that taught in, e.g. U.S.Pat. No. 6,222,030 to Dellinger et al.; U.S. Pat. Appl'n Publ'n No.US2002/0058802 A1 to Dellinger et al.; Seio et al. (2001) TetrahedronLett. 42 (49):8657-8660. These references describe two-step methods of(1) coupling a hydroxyl-protected nucleoside monomer to a growingoligonucleotide chain, and (2) deprotecting the product using an alphaeffect nucleophilic reagent that also oxidizes the internucleotidelinkage to give a phosphotriester bond. The coupling anddeprotection/oxidation steps are repeated as necessary to give anoligonucleotide having a desired sequence and length.

The invention further provides a method for synthesizingpolynucleotides. In an embodiment, the method comprises forming aninternucleotide bond by contacting a functionalized support having thestructure (Ic) with a precursor having the structure (IIc) as describedabove under conditions and for a time sufficient to allow the precursorto react with the nucleoside moiety of the functionalized support toresult in formation of the internucleotide bond. In particularembodiments, the method comprises forming an internucleotide bond bycontacting a functionalized support having the structure (Ic) with aprecursor having the structure (IVc) as described above under conditionsand for a time sufficient to allow the precursor to react with thenucleoside moiety of the functionalized support to result in formationof the internucleotide bond. The nucleoside moiety typically comprises areactive site hydroxyl that is capable of reacting with the reactivegroup of the precursor to result in formation of an internucleotidebond.

In an embodiment, the method of synthesizing polynucleotides furtherincludes, after the internucleotide bond is formed, exposing the resultof the forming an internucleotide bond step to a composition whichconcurrently oxidizes the internucleotide bond and removes ahydroxylprotecting group (the simultaneous deprotection and oxidationstep).

In an embodiment of the method for 3′ to 5′ synthesis, a precursorhaving the structure (IVc) as described above is contacted with anucleoside moiety of a functionalized support having the structure(VIIc):

Wherein the groups are defined as follows:

Base′ is a heterocyclic base, optionally protected with a protectinggroup

is a solid support, and

R1, Lnk′, Trl, Lnk, and (Ntd)_(k) are each as described above.

The nucleoside moiety of structure (VIIIc) has a reactive site hydroxylthat is available to react with the reactive group of a precursor. Thecoupling reaction is performed under conditions and for a timesufficient to allow the precursor to react with the nucleoside moiety toresult in formation of the internucleotide bond. The product of thecoupling reaction may be represented as structural formula (IXc), asfollows:

Wherein the groups are defined as follows:

is a solid support,

R^(hpg) represents a hydroxylprotecting group releasable underconditions of simultaneous deprotection and oxidation during thepolynucleotide synthesis cycle,

Each R1 is independently as described above with regard to structure(IVc),

Base, Base′, Tram, Y, Lnk′, Trl, Lnk, and (Ntd)_(k) are each asdescribed above.

In FIG. 3, this step (the “coupling” reaction) is illustrated in contextof a full synthesis cycle. The coupling reaction may be conducted understandard conditions used for the synthesis of oligonucleotides andconventionally employed with automated oligonucleotide synthesizers.Such methodology will be known to those skilled in the art and isdescribed in the pertinent texts and literature, e.g., in D. M. Matteuciet al. (1980) Tet. Lett. 521:719 and U.S. Pat. No. 4,500,707.

It will be apparent to the skilled reader that, for polynucleotidesynthesis, different bases may be employed each synthesis cycledepending on the desired sequence of the final product. Also, in somecycles, the desired heterocyclic base may not have an exocyclic amineand thus not require an exocyclic amine triaryl methyl protecting groupin accordance with the invention. Synthesis of polynucleotides havingsuch desired sequences is within the scope of the invention, as impliedby the Base′ group in structures (VIIIc) and (IXc).

In the second step of the synthesis cycle shown in FIG. 3, the productis treated with a combined deprotection/oxidation reagent to oxidize thenewly formed internucleotide bond and to remove the hydroxylprotectinggroup at the 5′ terminus, thus converting the moiety —OR^(hpg) to —OH.The resulting —OH is then available to serve as the reactive sitehydroxyl for the next round of the synthesis cycle. Advantageously, thecombined deprotection/oxidation step may be conducted in connection withfluorescent or other readily detectable hydroxylprotecting groups,enabling monitoring of individual reaction steps. Further, the method isuseful in carrying out either 3′-to-5′ synthesis or 5′-to-3′ synthesis.Finally, the method readily lends itself to the highly parallel,microscale synthesis of oligonucleotides.

The deprotection/oxidation reaction essentially may be conducted underthe reported conditions used for the synthesis of polynucleotides asdescribed in, e.g. U.S. Pat. No. 6,222,030 to Dellinger et al.; U.S.Pat. Appl'n Publ'n No. US2002/0058802 A1 to Dellinger et al.; Seio etal. (2001) Tetrahedron Lett. 42 (49):8657-8660. As will be appreciatedby those of ordinary skill in the art, given the disclosure herein, theconditions for the deprotection/oxidation step may vary depending on thenature of the protecting groups used. In order to be compatible with thetriaryl methyl linker group and the exocyclic amine triaryl methylprotecting group provided for by the current invention, the conditionsfor the simultaneous deprotection and oxidation step (i.e. requiredconditions for release of the hydroxylprotecting group) should beselected such that the nascent polynucleotide remains attached to thesoluble support via the triaryl methyl linker group, and the exocyclicamine triaryl methyl protecting groups remain stably attached to theexocyclic amine groups. Typical conditions for thedeprotection/oxidation reaction include a pH in the neutral tomoderately basic range. In particular embodiments, the pH of thedeprotection/oxidation reaction is at least about 6.0, typically atleast about 6.5, more typically at least about 7.0, still more typicallyat least about 7.5, and the pH is typically less than about 12,typically less than about 11, more typically less than about 10.5, stillmore typically less than about 10.

The combined deprotection/oxidation reagent may be selected to provideparticularly advantageous synthesis conditions and characteristics, asare described herein. In an embodiment, the combineddeprotection/oxidation reagent provides for contacting of the elongatingpolynucleotide chain with an alpha effect nucleophile under neutral ormildly basic aqueous conditions to remove reactive sitehydroxylprotecting groups where such protecting groups are labile undernucleophilic attack; the alpha effect nucleophile also serves to oxidizethe phosphite triester linkage to a phosphotriester linkage.

In an embodiment, the combined deprotection/oxidation reagent provides anucleophilic deprotection reagent under neutral or mildly basicconditions in aqueous solution. During the second step of thepolynucleotide synthesis cycle (the deprotection/oxidation step in FIG.3), the product is treated with an “alpha effect” nucleophile in orderto remove the protecting group at the reactive site hydroxyl (e.g. the5′ terminus), thus converting the moiety —OR^(hpg) to —OH. The alphaeffect nucleophile also oxidizes the newly formed phosphite triesterlinkage to give the phosphotriester linkage as shown in FIG. 3.

The deprotection/oxidation reagent may be any compound or mixture ofcompounds that is compatible with the synthesis of polynucleotides andhas the properties discussed herein. Typically, thedeprotection/oxidation reagent includes a concentration of an oxidantthat is high enough to rapidly oxidize the newly formed phosphiteinternucleotide linkage. This is typically at least 0.1% vol/vol,typically at least 0.5% vol/vol, more typically at least about 1.0%vol/vol, still more typically at least about 3.0% vol/vol. Theconcentration of the oxidant typically should be low enough to avoidappreciable (e.g. less than 1% per iteration of the synthesis cycle)amounts of oxidative destruction of the nucleobases or protectednucleobases. This concentration is typically less than 10% vol/vol, moretypically less than 9% vol/vol, still more typically less than 7%vol/vol.

The deprotection/oxidation reagent in typical embodiments provides asource of a peroxyanion at neutral to mildly basic pH in the reactionmixture during the deprotection/oxidation reaction. The concentration ofthe peroxyanion will be related to the acid dissociation constant of thehydroperoxide species at equilibrium. The concentration of peroxyanionis typically in the range 0.01% to 99% of the total hydroperoxideconcentration (i.e. sum of all hydroperoxide species, e.g. protonatedand unprotonated forms), more typically in the range 0.05% to 90% of thetotal hydroperoxide concentration, yet more typically in the range 0.1%to 50% of the total hydroperoxide concentration, still more typically ina range of 1.0% to 30% of the total hydroperoxide concentration.

In certain embodiments, the nucleophilic deprotection reagent thatexhibits an alpha effect is a peroxide or a mixture of peroxides. Intypical embodiments, the pH at which the deprotection/oxidation reactionis conducted is generally in the range of about three pH units below thepKa of the nucleophilic deprotection reagent (that is, the pKa forformation of the corresponding peroxy anion) up to about three pH unitsabove the pKa of the nucleophilic deprotection reagent. More typically,the pH of the deprotection/oxidation reaction is in the range of aboutone pH unit below the pKa of the nucleophilic deprotection reagent up toabout pH 11. Preferably the pH will be the range that allows a highenough concentration of the peroxy anion to form, e.g. from about thepKa of the peroxide up to a pH of about 11. The peroxide may be eitherinorganic or organic. Suitable inorganic peroxides include those of theformula M+OOH—, where M+ is any counter ion, including for example H+,Li+, Na+, K+, Rb+, Cs+, or the like; and lithium peroxide or hydrogenperoxide and alkaline stabilized forms thereof can be particularlysuitable. Suitable organic peroxides include those of the formula ROOH,where R is selected from the group consisting of alkyl, aryl,substituted alkyl, substituted aryl, and modified alkyl. Moreparticularly, the organic peroxide will have one of the following threegeneral structures (Xc), (XIc) or (XIIc)

in which R21 through R27 are generally hydrocarbyl optionallysubstituted with one or more nonhydrocarbyl substituents and optionallycontaining one or more nonhydrocarbyl linkages. Generally, R21 throughR27 are independently selected from the group consisting of hydrido,alkyl, modified alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,alkenyl, cycloalkenyl, alkynyl aralkynyl, cycloalkynyl, substitutedaralkyl, substituted cycloalkyl, substituted cycloalkylalkyl,substituted alkenyl, substituted cycloalkenyl, substituted alkynylsubstituted aralkynyl, substituted cycloalkynyl, hydrocarbyl, andsubstituted hydrocarbyl. T-butyl-hydroperoxide ormetachloroperoxybenzoic acid can be particularly suitable. As a specificexample, the m-chloroperoxybenzoic acid (mCPBA) peroxy anion has beenfound to be useful for removal of protecting groups on the reactive sitehydroxyl.

As indicated in FIG. 3, the steps of the synthesis cycle include acoupling step and a simultaneous deprotection/oxidation step. In anembodiment of a method of synthesizing a polynucleotide in accordancewith the present invention, these steps of the synthesis cycle may berepeated multiple times to produce a polynucleotide having the desiredsequence.

The use of a triaryl methyl protecting group provides for protection ofthe exocyclic amines from undesirable side reactions during synthesis.The triaryl methyl protecting groups may then be removed from thesynthesized polynucleotide under mildly acidic conditions. In particularembodiments, the triaryl methyl protecting groups may be removed by weakacids under conditions that do not result in destruction of theglycosidic linkage, typically glacial acetic acid or glacial aceticacid/water mixtures.

In typical embodiments, the synthesized polynucleotide may be releasedfrom the solid support to yield the polynucleotide free in solution (notattached to the support). This reaction typically is conducted undermildly acidic conditions. In particular embodiments, the synthesizedpolynucleotide is cleaved from the triaryl methyl linker group by weakacids under conditions that do not result in destruction of theglycosidic linkage, typically glacial acetic acid or glacial aceticacid/water mixtures. In certain embodiments, the reaction to release thepolynucleotides from the solid support also results in removal of thetriaryl methyl protecting groups (i.e. the reactions are performedconcurrently).

The functionalized support which is contacted with the precursor in themethod of the present invention typically comprises a nucleoside moietybound to a solid support directly or via an intervening polynucleotidestrand. The solid support may comprise any suitable material adapted forits intended use in polynucleotide synthesis. The solid support shouldbe essentially inert to the conditions of reactions used for thepolynucleotide synthesis. Typically the solid support has a surface towhich the nucleoside moiety (or a polynucleotide which comprises thenucleoside moiety) is bound, directly or indirectly (i.e. via anintermediate moiety or moieties, e.g. moieties typically referred to inthe art variously as linking groups, tethers, or spacers); such that thenucleoside moiety, which has the reactive site hydroxyl, is accessibleto the precursor when the functionalized support is contacted with asolution containing the precursor.

In certain embodiments, the solid support comprises a solid substrateand a modification layer disposed on or bound to (directly orindirectly) the substrate, and the nucleoside moiety having the reactivesite hydroxyl is bound to (directly or indirectly) the modificationlayer. Such modification layer may be formed on the substrate by methodsknown in the art of modifying surface properties of supports used inpolynucleotide synthesis, or known in the art of modifying supports toprovide desired surface properties. In certain embodiments, themodification layer may be, e.g., a coating, a material deposited bydeposition techniques known in the art, a hydrophobic layer, or ahydrophilic layer. In particular embodiments, the support comprises achemically active group bound to a substrate via a silane group.Particularly contemplated are solid supports taught in U.S. Pat. No.6,258,454 to Lefkowitz et al. (2001) as solid supports having achemically active moiety bound to a substrate via a linking groupattached to a silane group bound to the surface of a substrate.

EXAMPLES

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of synthetic organic chemistry,biochemistry, molecular biology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions disclosed and claimedherein. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, percents are wt./wt., temperature is in ° C. andpressure is at or near atmospheric. Standard temperature and pressureare defined as 20° C. and 1 atmosphere.

A synthesis of reagents used in certain embodiments of the presentinvention is now described. It will be readily apparent that thereactions described herein may be altered, e.g. by using modifiedstarting materials to provide correspondingly modified products, andthat such alteration is within ordinary skill in the art. Given thedisclosure herein, one of ordinary skill will be able to practicevariations that are encompassed by the description herein without undueexperimentation.

Abbreviations used in the examples include: THF is tetrahydrofuran; TLCis thin layer chromatography; HEX is hexane; Et₃N is triethylamine; MWis molecular weight; AcCN is acetonitrile; sat'd is saturated; EtOH isethanol; B is a heterocyclic base having an exocyclic amine group,B^(Prot) is a heterocyclic base having an exocyclic amine group with atrityl protecting group on the exocyclic amine group; TiPSCl is1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane; TEMED isN,N,N′,N′-Tetramethylethylenediamine; Py is pyridine; MeCN isacetonitrile; DMT is dimethoxytrityl; MMT is monomethoxytrityl; TMT istrimethoxytrityl; Cyt^(DMT) is cytosine which has a dimethoxytritylprotecting group on the exocyclic amine group; Cyt^(TMT) is cytosinewhich has a trimethoxytrityl protecting group on the exocyclic aminegroup (and so on for other bases and protecting groups on the exocyclicamine group of the indicated base); MS is mass spectrometry, MS (ES) ismass spectrometry (electrospray), HRMS (FAB) is high resolution massspectrometry (fast atom bombardment); DCM is methylene chloride; EtOAcis ethyl acetate; ^(i)Pr is isopropyl; Et₃N is triethylamine; TCA istrichloroacetic acid; TEAB is tetraethylammonium bicarbonate. Tritylrefers to a substituted or unsubstituted triphenyl methyl group; contextmay indicate which is desired or intended.

Example A5′-O-(4-(3-(4-Nitrophenoxycarbonyl)-aminopropoxy)-4′-methoxytrityl)-2′-deoxythymidineis produced by the following protocol 4-HYDROXY-4′-METHOXYTRITYL ALCOHOLSTEP 1

-   -   A. 25.0 g (126.2 mmoles) 4-hydroxy Benzophenone (1); Aldrich #        H2020-2    -   B. 500 ml THF; Aldrich # 49446-1    -   C. 700 ml of a 0.5 M Solution in THF (175 mmoles) 4-Anisyl        Magnesium Bromide; Alpha-Aesar # 89435

TLC System: HEX/EtOAc/Acetone (4:1:1)+0.5% Et₃N on silica gel.

Using a 3-L 3-neck round bottom flask with a mechanical stirrer, U-tubethermometer and drying tube, (A) was added to (B) and the solution wascooled to 4° C. in a dry-ice/acetone bath, under Argon atmosphere. (C)was added drop wise over a period of 1 hour. Precipitate forms tan→pinkcolor. The temperature was kept between 0-5° C. during the addition. Themixture was removed from the bath and stirred at ambient temperature(under Argon atmosphere) for 16-hours. The solvent was evaporated invacuo. The residue was suspended in 300 ml ether and 200 mL cold water.The ether layer was extracted with 150 mL saturated NaHCO₃ and 150 mLsaturated NaCl and dried with MgSO₄. The solvent was evaporated, and 66g of an oily residue was obtained. The residue was dissolved in 50 mLDCM, 30 g silica gel added and column purified over silica gel, withDCM/AcCN (19:1) as the initial mobile phase, changing to DCM/AcCN (9:1)as mobile phase for elution of the product. The product was columnpurified a second time over silica gel using EtOAc/HEX (1:1) as mobilephase for elution of the product.

Theoretical Yield: 38.6 g Actual Yield: 23.9 g [62%]¹H NMR (CDCl₃) 3.78(3H, s), 6.75 (2H, d, J=8.8), 6.83 (2H, d, J=8.8), 7.11 (2H, d, J=8.8),7.17 (2H, d, J=8.8), 7.25-7.32 (5H, m); MS (ESI−) m/z 305 (M−1, 100);(ESI+) m/z 635 (M₂+Na, 33), 289 (M−H₂O, 100)

4-(3-PHTHALIMIDOPROPOXY)-4′-METHOXYTRITYL ALCOHOL STEP 2

TLC System: DCM/AcCN [19:1]

-   -   A. 24.0 g (78.0 mmoles) [2]    -   B. 21.6 g (156 mmoles) potassium carbonate MW=138.1; Aldrich #        20961-9    -   C. 63 g (235 mmoles) 3-bromopropyl phthalamide MW=268.1; Aldrich        # B8000-3    -   D. Single (Dry) Crystal Potassium Iodide MN 166.1; Aldrich #        22194-5    -   E. 600 mL Toluene

Using a 2 L 3-neck round bottom flask equipped with a thermometer,reflux condenser, drying tube and stir bar, (A), (B) (C), and (D) wereadded to (E) in sequential order. The mixture was heated to reflux for24 hours. The solvent was evaporated. The residue was partitionedbetween 750 mL DCM and 300 mL water. The DCM layer was washed twice with400 mL sat'd NaCl then dried over MgSO₄. The product was then columnpurified over a silica gel column packed with DCM and eluted withDCM/AcCN (19:1) as mobile phase.

Theoretical Yield: 38.5 g Actual Yield: 20 g [52%]¹H NMR (CDCl₃)2.14-2.18 (2H, m), 2.91 (1H, s), 3.78 (3H, s), 3.87 (2H, t, J=7.0), 3.99(2H, t, J=6.2), 6.71 (2H, d, J=8.8), 6.80 (2H, d, J=8.8), 7.10 (2H, d,J=8.8), 7.15 (2H, d, J=8.8), 7.23-7.28 (5H, m), 7.66-7.69 (2H, m),7.79-7.81 (2H, m); ¹³C NMR 28.3, 35.4, 55.2, 65.6, 37.8, 113.0, 113.6,123.2, 126.9, 127.7, 129.0, 129.1, 132.0, 133.9, 139.4, 139.5, 147.3,157.7, 158.5, 168.3; MS (FAB+) m/z 493 (M, 25), 476 (M−OH, 100).

4-(3-AMINOPROPOXY)-4′-METHOXYTRITYL ALCOHOL STEP 3

-   -   A. 17.0 g (34.4 mmoles) [3]    -   B. 17.3 mL (340 mmoles) Hydrazine monohydrate ME=50; Aldrich #        20794-2    -   C. 420 mL EtOH

TLC System: Acetone/Methanol [3:2]

Using a 1 L 1-neck round bottom flask equipped with a reflux condenser,(A) was dissolved in (C) and (B) was added. The mixture was heated toreflux for 1 hour. The precipitate was filtered and the solvent wasevaporated. The residue was partitioned between ether and water. Theether layer was separated and the solvent evaporated in vacuo. Theresidue was azeotroped twice with 200 mL of methanol. TLC analysisshowed a single spot. The material was moved to step 4 without furtherpurification.

Theoretical Yield: 12.6 g Actual Yield 14.2 g [100%]¹H NMR (CDCl₃)1.79-1.86 (2H, m), 2.20 (1H, br s), 2.77 (2H, t, J=7.0), 3.75 (3H, s),3.95 (2H, t, J=6.2), 6.77-6.80 (4H, m), 7.14-7.18 (4H, m), 7.20-7.27(5H, m); ¹³C NMR (CDCl₃) 32.6, 39.0, 55.1, 57.8, 65.7, 81.0, 112.9,113.4, 126.8, 127.6, 127.7, 129.1, 139.7, 147.5, 157.7, 158.3; MS (ESI−)m/z 362 (M−1, 100); (ESI+) m/z 364 (M+1, 100), 727 (M₂+1, 37).

4-(3-(4-NITROPHENOXYCARBOXY)AMINOPROPOXY)-4′-METHOXY-TRITYL ALCOHOL STEP4

-   -   A. 7.5 g (20.5 mmoles) [4]    -   B. 6.25 g (20.5 mmoles) bis-4Nitrophenyl carbonate MW=304.21;        Aldrich # 16169-1    -   C. 450 mL DCM

TLC System: Acetone/Methanol [9:1]

Using a 1 L 1-neck round bottom flask, (A) was dissolved in (C) and (B)was added. The mixture was stirred for 24 hours at ambient temperature.TLC showed completion by spot to spot conversion of the startingmaterial. Extract reaction mixture with 4×150 mL saturated NaHCO₃, 2×200mL half-saturated NaCl and dried over MgSO₄. The product was then columnpurified over a silica gel column packed with HEX/EtOAc (2:1) and elutedwith HEX/EtOAc (1:1) as mobile phase. The product was column purified asecond time over silica gel using HEX/EtOAc/DCM (2:1:1) as mobile phasefor elution of the product.

Theoretical Yield: 10.8 g Actual Yield 7.0 g [65%]¹H NMR (CDCl₃)2.02-2.08 (2H, m), 3.05 (1H, br s), 3.45-3.53 (2H, m), 3.77 (3H, s),4.05 (2H, t, J=5.6), 5.70 (1H, t, J=5.6), 6.79-6.82 (4H, m), 7.13-7.17(4H, m), 7.23-7.30 (7H, m), 8.18 (2H, d, J=9.1); ¹³C NMR (CDCl₃) 28.8,39.1, 55.1, 65.8, 81.3, 113.1, 113.6, 121.9, 125.0, 127.0, 127.7, 127.8,129.1, 129.2, 139.3, 139.9, 144.6, 147.2, 153.1, 155.8, 157.5, 158.6; MS(FAB+) m/z 528 (M, 28), 511 (M−OH, 100).

4-(3-(4-NITROPHENOXYCARBOXY)AMINOPROPOXY)-4′-METHOXY-TRITYL CHLORIDESTEP 5

TLC System: Hexane/EtOAc [2:1]

-   -   A. 3.50 g (6.63 mmol) [5]    -   B. 11.6 mL (133 mmol) oxalyl chloride MW=126.9; Aldrich #        32042-0    -   C. 120 mL Hexane

A 250 mL 3-neck round bottom flask was equipped with a cold-fingerreflux/distillation condenser, magnetic stir bar, and two silicon rubbersepta. (A) was suspended in (C) in the flask, and the flask was placedunder argon and stirred. (B) was added to the stirring solution dropwise. Upon addition the suspended material dissolved and small bubblesformed in the flask. The reaction was refluxed overnight. The nextmorning the refluxing reaction consisted of a clear refluxing solutionand a viscous orange-red oil on the bottom of the flask. The condenserwas then set to distill and the hexanes and excess (B) removed bydistillation. The remaining oil was placed under high vacuum resultingin 6.7 g of a foamed solid, used in the following reaction.

Theoretical Yield: 6.6 g Actual Yield 6.7 g [100%]

5′-O-(4-(3-(4-NITROPHENOXYCARBOXY)AMINOPROPOXY)-4′-METHOXYTRITYL)-2′-DEOXYTHYMIDINESTEP 6

The foamed solid was then reacted with thymidine, in anhydrous pyridine,to give spot to spot conversion to the5′-O-(4-(3-(4-Nitrophenoxycarbonyl)aminopropoxy),4′-methoxytrityl)-2′-deoxythymidine product, shown in structure (XIIIc).

5′-O-(4-(3-(4-Nitrophenoxycarbonyl)-aminopropoxy)-4′-methoxytrityl)-2′-deoxythymidine

¹H NMR (CDCl₃) 1.47 (3H, s), 2.05-2.16 (2H, m), 2.26-2.55 (2H, m),3.35-3.53 (5H, m), 3.78 (3H, s), 4.02-4.10 (3H, m), 4.58 (1H, br s),5.88 (1H, t, J=5.9), 6.40 (1H, t, J=7.0), 6.80-6.85 (4H, m), 7.21-7.27(9H, m), 7.40 (2H, d, J=7.4), 7.59 (1H, s), 8.22 (2H, d, J=8.9), 9.90(1H, s); ¹³C NMR (CDCl₃); 11.7, 28.9, 38.9, 40.8, 53.4, 55.2, 63.6,65.8, 72.2, 84.8, 86.3, 86.8, 111.1, 113.2, 113.7, 121.9, 124.9, 127.1,127.9, 128.0, 129.9, 130.0, 135.1, 135.9, 144.2, 144.6, 150.6, 153.2,155.9, 157.6, 158.6, 164.1; MS (FAB+) m/z 753 (M+1, 100).

It will be apparent to one of skill in the art that the series ofsyntheses described above may be altered to employ analogous startingmaterials that react in a similar manner to give analogous products, andthat such alteration of the synthesis is within ordinary skill in theart. For example, in the last step, thymidine may be replaced withN-4-dimethoxy trityl-2′-deoxycytidine to give5′-O-(4-(3-(4-Nitrophenoxycarbonyl)-aminopropoxy)-4′-methoxytrityl)-N-4-dimethoxytrityl-2′-deoxycytidineas the final product. As another example, in step 2, the 3-bromopropylphthalamide may be replaced with 2-bromo ethyl phthalamide to give4-(3-phthalamidoethoxy)-4′-methoxytrityl alcohol as the product of step2. As another example, it will be appreciated that the nucleoside moietymay be bound to the triaryl methyl linker group via either the 3′-OH orthe 5′-OH. Such a modification will be accomplished by reacting a5′-O-protected nucleoside with the trityl linker under conditions thatenhance the rate of trityl reaction with secondary hydroxyls such as theaddition of an acylation catalyst like N,N-dimethlyaminopyridine orsilver salts as well as other techniques well know to one skilled in theart.

Furthermore, in the reaction designated as “Step 1”, above, the startingmaterials may be modified to yield a product wherein one or more of thephenyl (or substituted phenyl) rings is replaced by an alternatearomatic ring moiety, such as substituted or unsubstituted aromaticgroups such as phenyl, biphenyl, naphthanyl, indolyl, pyridinyl,pyrrolyl, thiophenyl, furanyl, annulenyl, quinolinyl, anthracenyl, andthe like. Such products may then be used as alternative startingmaterials in the reaction designated “Step 2” (and so on through therest of the described syntheses) to give a triaryl methyl-modifiednucleotide monomer, above.

As shown in the reaction designated (XIVc), below, the 5′-linkedmolecules can then be reacted with a support having a reactive moietysuch as amine or hydroxyl group, wherein the support is suitable for usefor polynucleotide synthesis.

The 3′-hydroxyl of the nucleoside moiety may then be used as a startingpoint for performing rounds of a polynucleotide synthesis reaction togive a product in which a polynucleotide strand is bound to thesubstrate via the trityl group. Once the synthesis is complete, thepolynucleotide can be released from the support with mild acid.

Example B 4-Hydroxy-4′-Methoxytrityl Alcohol Step 1

-   -   A. 25.0 g (126.2 mmoles) 4-hydroxy Benzophenone (1); Aldrich #        H2020-2    -   B. 500 ml THF; Aldrich # 49446-1    -   C. 700 ml of a 0.5 M Solution in THF (175 mmoles) 4-Anisyl        Magnesium Bromide; Alpha-Aesar # 89435

TLC System: HEX/EtOAc/Acetone (4:1:1)+0.5% Et₃N on silica gel

Using a 3-L 3-neck round bottom flask with a mechanical stirrer, U-tubethermometer and drying tube, (A) was added to (B) and the solution wascooled to 4° C. in a dry-ice/acetone bath, under Argon atmosphere. (C)was added drop wise over a period of 1 hour. Precipitate forms tan→pinkcolor. The temperature was kept between 0-5° C. during the addition. Themixture was removed from the bath and stirred at ambient temperature(under Argon atmosphere) for 16-hours. The solvent was evaporated invacuo. The residue was suspended in 300 ml ether and 200 mL cold water.The ether layer was extracted with 150 mL saturated NaHCO₃ and 150 mLsaturated NaCl and dried with MgSO₄. The solvent was evaporated, and 66g of an oily residue was obtained. The residue was dissolved in 50 mLDCM, 30 g silica gel added and column purified over silica gel, withDCM/AcCN (19:1) as the initial mobile phase, changing to DCM/AcCN (9:1)as mobile phase for elution of the product. The product was columnpurified a second time over silica gel using EtOAc/HEX (1:1) as mobilephase for elution of the product.

Theoretical Yield: 38.6 g Actual Yield: 23.9 g [62%]¹H NMR (CDCl₃) 3.78(3H, s), 6.75 (2H, d, J=8.8), 6.83 (2H, d, J=8.8), 7.11 (2H, d, J=8.8),7.17 (2H, d, J=8.8), 7.25-7.32 (5H, m); MS (ESI−) m/z 305 (M−1, 100);(ESI+) m/z 635 (M₂+Na, 33), 289 (M−H₂O, 100).

4-((3-Propoxy)-tert-Butyldimethylsilane)-4′-Methoxytrityl Alcohol Step 2

TLC System: DCM/AcCN [19:1]

-   -   A. 24.0 g (78.0 mmoles) [2]    -   B. 21.6 g (156 mmoles) potassium carbonate MW=138.1; Aldrich #        20961-9    -   C. 60 g (235 mmoles) (3-Bromopropoxy)-tert-butyldimethylsilane        MW=253.3; Aldrich # 42,906-6    -   D. Single (Dry) Crystal Potassium Iodide MN 166.1; Aldrich #        22194-5    -   E. 600 mL Toluene

Using a 2 L 3-neck round bottom flask equipped with a thermometer,reflux condenser, drying tube and stir bar, (A), (B) (C), and (D) wereadded to (E) in sequential order. The mixture was heated to reflux for24 hours. The solvent was evaporated. The residue was partitionedbetween 750 mL DCM and 300 mL water. The DCM layer was washed twice with400 mL sat'd NaCl then dried over MgSO₄ to yield the4-((3-Propoxy)-tert-Butyldimethylsilane)-4′-Methoxytrityl Alcohol.

Theoretical Yield: 37.3 g Actual Yield: 16 g [43%]MS (FAB+) m/z 479, 462(M−OH, 100).

4-((3-Propoxy)-tert-Butyldimethylsilane)-4′-Methoxytrityl Chloride Step3

TLC System: Hexane/EtOAc [2:1]

-   -   A. 5.0 g (110.44 mmol) [7]    -   B. 18.2 mL (208 mmol) oxalyl chloride MW=126.9; Aldrich #32042-0    -   C. 150 mL Hexane

A 250 mL 3-neck round bottom flask was equipped with a cold-fingerreflux/distillation condenser, magnetic stir bar, and two silicon rubbersepta. (A) was suspended in (C) in the flask, and the flask was placedunder argon and stirred. (B) was added to the stirring solution dropwise. Upon addition the suspended material dissolved and small bubblesformed in the flask. The reaction was refluxed overnight. The nextmorning the refluxing reaction consisted of a clear refluxing solutionand a viscous orange-red oil on the bottom of the flask. The condenserwas then set to distill and the hexanes and excess (B) removed bydistillation. The remaining oil was placed under high vacuum resultingin 6.7 g of a foamed solid, used in the following reaction.

Theoretical Yield: 5.2 g Actual Yield 5.2 g [100%]

3′-O-(4-Chlorophenyl)-Carbonyl-2′-Deoxythymidine

5′-O-(4,4′-Dimethoxytrityl)-2′-deoxythymidine (10.89 g, 20.0 mmol) wascoevaporated from pyridine (3×40 mL), dissolved in pyridine (180 mL),and 4-chlorophenyl chloroformate (3.06 mL, 24.0 mmol) added withvigorous stirring. The mixture was stirred for 2 hours, solvent removedin vacuo, and the oily residue coevaporated with toluene (100 mL). Theresulting oil was dissolved in dichloromethane (500 mL), extracted withsaturated NaHCO₃ (250 mL) and brine (250 mL), dried over MgSO₄, andsolvent evaporated to yield a viscous yellow oil. Purification by silicagel chromatography (0-2% ethanol in100:0.1dichloromethane:triethylamine) yielded3′-O-(4-chlorophenyl)-carbonyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidineas a white, glassy solid (10.93 g, 78.2%).

Anal. ¹H NMR (400 MHz, CDCl₃) δ 9.27 (1H, s, H₃), 7.63 (1H, s, H₆),6.85-7.42 (17H, m), 6.54 (1H, m, H_(1′)), 5.43 (1H, m, H_(3′)), 4.32(1H, m, H_(4′)), 3.78 (6H, s), 3.44-3.59 (2H, m, H_(5′)), 2.47-2.68 (2H,m, H_(5′,5″)), 1.40 (3H, s); ¹³C NMR (100.5 MHz, CDCl₃) δ 163.7, 158.8,152.7, 149.7, 149.2, 144.1, 135.1, 135.0, 131.7, 130.1, 130.0, 129.8,129.6, 128.0, 127.2, 122.2, 113.3, 111.7, 87.3, 84.3, 83.6, 79.9, 63.6,55.2, 37.8, 11.6; MS (FAB+) m/z 698 (M, 100).

To3′-O-(4-chlorophenyl)-carbonyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine(2.50 g, 3.58 mmol) was added a 3% solution of trichloroacetic acid indichloromethane (400 mL) with vigorous stirring. The mixture was stirredfor 3 min before pyridine/methanol (1:1) was added drop wise until thered color of the DMT cation was quenched. The mixture was extracted withsaturated NaHCO₃ (300 mL) and brine (300 mL), dried over MgSO₄, andsolvent removed in vacuo. Purification of the resulting oil by silicagel chromatography (0-6% ethanol in dichloromethane) afforded the3′-O-(4-Chlorophenyl)-Carbonyl-2′-Deoxythymidine.as a white powder (1.30g, 92%);

Anal. Calcd. for C₁₇H₁₇ClN₂O₇: C, 51.5; H, 4.3; N, 7.1. Found: C, 51.3;H, 4.5; N, 7.0. ¹H NMR (400 MHz, CDCl₃/d₄-MeOH) 9.57 (1H, s, H₃), 7.44(1H, s, H₆), 7.25 (2H, d, J=8.8), 7.03 (2H, d, J=8.8), 6.17 (1H, m,H_(1′)), 5.27 (1H, m, H_(3′)), 4.17 (1H, m, H_(4′)), 3.83 (2H, m,H_(5′)), 2.42 (2H, m, H_(2′,2″)), 1.80 (3H, s); ¹³C NMR (100.5 MHz,CDCl₃/d4-MeOH) 164.1, 152.8, 150.6, 149.2, 136.7, 131.7, 129.6, 122.2,111.4, 86.3, 84.8, 79.5, 62.4, 37.0, 12.5; MS (ESI+) m/z 397 (M+1, 100).

5′-O-4-((3-Propoxy)-tert-Butyldimethylsilane)-4″-Methoxytrityl-3′-O-(4-Chlorophenyl)-Carbonyl-2′-DeoxythymidineStep 4

To 3′-O-(4-chlorophenyl)-carbonyl-2′-deoxythymidine (1.2 g, 3.1 mmol) inpyridine (35 mL) was added4-((3-Propoxy)-tert-Butyldimethylsilane)-4′-Methoxytrityl Chloride (1.86g, 3.75 mmol). The mixture was stirred for 4 h at which point thesolvent was removed under reduced pressure. The residue was dissolved indichloromethane, washed with 5% sodium carbonate and brine, dried(MgSO₄), and solvent removed in vacuo to yield a pale yellow oil. The5′-O-4-((3-Propoxy)-tert-Butyldimethylsilane)-4″-Methoxytrityl-3′-O-(4-Chlorophenyl)-Carbonyl-2′-Deoxythymidinewas isolated by silica gel chromatography using 1-4%methanol/dichloromethane as eluant as a pale yellow glassy solid (2.4 g,90.0%); MS (FAB+) m/z 743 (M, 100).

5′-O-4-(3-Hydroxypropyl)-4′-Methoxytrityl-3′-O-(4-Chlorophenyl)-Carbonyl-2′-DeoxythymidineStep 5

5′-O-4-((3-Propoxy)-tert-butyldimethylsilane)-4″-methoxytrityl-3′-O-(4-chlorophenyl)-carbonyl-2′-deoxythymidine(2.4 g, 2.8 mmol) was dissolved in anhydrous pyridine (75 mL) using amagnetic stirrer. The flask was kept anhydrous under argon and cooled inan ice/water bath. Hydrogen fluoride pyridine (100 μL) Fluka cat# 47586was dissolved in 10 mL of anhydrous pyridine and added to the stirringflask. The reaction was allowed to stir for 30 min then evaporated to arust brown oil. The residue was dissolved in dichloromethane, washedwith 5% sodium carbonate and brine, dried (MgSO₄), and solvent removedin vacuo to yield a dark yellow oil. The5′-O-4-(3-Hydroxypropyl)-4″-Methoxytrityl-3′-O-(4-Chlorophenyl)-Carbonyl-2′-Deoxythymidinewas isolated by silica gel chromatography using 0-3%methanol/dichloromethane as eluant as a pale yellow glassy solid (2.4 g,90.0%); MS (FAB+) m/z 859 (M, 100).

5′-O-4-(3-propyloxy(2-CyanoethylN,N-diisopropylphosphoramidite))-4″-Methoxytrityl-3′-O-(4-Chlorophenyl)-Carbonyl-2′-DeoxythymidineStep 6

5′-O-4-(3-Hydroxypropyl)-4″-Methoxytrityl-3′-O-(4-Chlorophenyl)-Carbonyl-2′-Deoxythymidine3.7 g (5.0 mmol) and tetrazole (175 mg, 2.50 mmol) were dried undervacuum for 24 h then dissolved in dichloromethane (100 mL). 2-CyanoethylN,N,N′,N′-tetraisopropylphosphorodiamidite (2.06 mL, 6.50 mmol) wasadded in one portion and the mixture stirred over 1 h. The reactionmixture was washed with sat. NaHCO₃ (150 mL) and brine (150 mL), driedover MgSO₄, and applied directly to the top of a silica columnequilibrated with hexanes. The dichloromethane was flashed off thecolumn with hexanes, and the product eluted as a mixture ofdiastereoisomers using 1:1 hexanes:ethyl acetate then ethyl acetate.After evaporation of solvents in vacuo and coevaporation withdichloromethane, product was isolated as friable, white, glassy solidsin 75% yield; ³¹P NMR (162.0 MHz, CDCl₃) 148.89, 148.85; MS (FAB+) m/z945 (FAB−) m/z 943

It will be apparent to one of skill in the art that the series ofsyntheses described above may be altered to employ analogous startingmaterials that react in a similar manner to give analogous products, andthat such alteration of the synthesis is within ordinary skill in theart. For example, thymidine may be replaced with N-4-dimethoxytrityl-2′-deoxycytidine in step 4 to give5′-O-4-(3-propyloxy-(2-cyanoethylN,N-diisopropyl-phosphoramidite))-4″-methoxytrityl-3′-O-(4-chlorophenyl)-carbonyl-N-4-dimethoxytrityl-2′-deoxycytidineas the final product. As another example, in step 2, the(3-bromopropoxy)-tert-butyldimethylsilane may be replaced with(4-bromobutoxy)-tert-butyldimethylsilane to give4-((4-Butoxy)-tert-butyldimethylsilane)-4′-methoxytrityl alcohol theproduct of step 2. As another example, it will be appreciated that thenucleoside moiety may be bound to the triaryl methyl linker group viaeither the 3′-OH or the 5′-OH. Such a modification will be accomplishedby reacting a 5′-O-protected nucleoside with the trityl linker underconditions that enhance the rate of trityl reaction with secondaryhydroxyls such as the addition of an acylation catalyst likeN,N-dimethlyaminopyridine or silver salts as well as other techniqueswell known to one skilled in the art.

Furthermore, in the reaction designated as “Step 1”, above, the startingmaterials may be modified to yield a product wherein one or more of thephenyl (or substituted phenyl) rings is replaced by an alternatearomatic ring moiety, such as substituted or unsubstituted aromaticgroups such as phenyl, biphenyl, naphthanyl, indolyl, pyridinyl,pyrrolyl, thiophenyl, furanyl, annulenyl, quinolinyl, anthracenyl, andthe like. Such products may then be used as alternative startingmaterials in the reaction designated “Step 2” (and so on through therest of the described syntheses) to give a triaryl methyl-modifiednucleotide monomer, above.

As shown in the reaction designated (XVc), below, the 5′-linkedmolecules can then be reacted with a support having a reactive moietysuch as a hydroxyl group, thiol group, or amino group, wherein thesupport is suitable for use for polynucleotide synthesis.

The 3′-hydroxyl of the nucleoside moiety may then be used as a startingpoint for performing cycles of a polynucleotide synthesis reaction togive a product in which a polynucleotide strand is bound to thesubstrate via the trityl group.

Preparation of 2-Furanyl-di-(2,4-dimethoxyphenyl)methanol

Methyl-2-furoate (1.58 g, 0.0125 mol) was dissolved in 100 mL of dryTHF. A dropping funnel containing 50 mL of a 0.5 M solution of2,4-dimethoxyphenylmagnesium bromide in THF (Aldrich) was attached tothe reaction flask and the system was purged with argon. The reactionflask was submerged in an ice bath for 5 minutes, at which time theGrignard solution was added drop-wise over 15 minutes. After additionwas complete, the reaction was allowed to warm to room temperature andwas stirred overnight. A brown solution resulted after 14 hours ofstirring. This solution was added to 150 g crushed ice and the slush wasstirred with ˜0.5 g sodium bicarbonate during drop-wise addition ofconc. HCl. A violet color was observed upon addition of acid, which wascontinued until effervescence was observed. The resulting neutralsuspension was extracted into ˜400 mL EtOAc and the organic layer wasdried over MgSO₄. Removal of solvent in a rotary evaporator gave aclear/yellow oil. Upon standing in ˜30 mL EtOAc (12 h), the oil hadturned deep blue and a white crystalline solid had settled out. Thesolid was collected and gave 1.7 g desired product. The blue solutionwas chromatographed in 50% EtOAc/50% hexanes and the second product toelute (deep violet upon acid treatment on TLC plate) was collected.Recrystallization of this product gave an additional 1.3 g pure desiredalcohol. 3.0 g/4.6 g=65% yield.

Preparation of 2-Furanyl-di-(2-methoxyphenyl)methanol

Methyl-2-furoate (1.58 g, 0.0125 mol) was dissolved in 100 mL of dryTHF. A dropping funnel containing 50 mL of a 0.5 M solution of2-methoxyphenylmagnesium bromide in THF (Aldrich) was attached to thereaction flask and the system was purged with argon. The reaction flaskwas submerged in an ice bath for 5 minutes, at which time the Grignardsolution was added drop-wise over 15 minutes. After addition wascomplete, the reaction was allowed to warm to room temperature and wasstirred overnight. A brown solution resulted. This solution was added to150 g crushed ice and the slush was stirred with ˜0.5 g sodiumbicarbonate during drop-wise addition of conc. HCl. A lavender color wasobserved upon addition of acid, which was continued until effervescencewas observed. The resulting neutral suspension was extracted into ˜400mL EtOAc and the organic layer was dried over MgSO₄. Removal of solventin vacuo gave a clear/yellow oil. The oil was purified by silica gelcolumn chromatography and the product eluted using a 50:50 (vol:vol)mixture of EtOAc:hexanes. The purified product was evaporated to a solidgum yielding 3.1 grams/82% yield. The product was characterized by HPLC,¹H NMR, and FAB mass spectroscopy FAB+311 m/e.

The compounds prepared in the previous two paragraphs are triaryl methylcompounds that are useful in preparing protected heterocyclic bases,e.g. by placing a triaryl methyl protecting group on exocyclic aminespresent on a desired heterocyclic base. Use of such compounds will bereadily apparent to persons of ordinary skill in the art given thedisclosure herein.

Trityl Protection of Nucleobases

Synthesis of 5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl)deoxynucleosides

Deoxynucleoside (10 mmole) was coevaporated 3 times with pyridine.Anhydrous pyridine (35 mL) and1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (3.47 g, 11 mmole) wereadded, and the mixture was stirred overnight at room temperature. Thesolution was concentrated and chromatographed on silica gel with 6%methanol in CHCl₃.

-   B=Cytosine: yield 99.9%; MS (ES) m/z 470(M+H)-   B=Adenine: yield 92%; MS (ES) m/z 494(M+H)-   B=Guanine; yield 98%; MS (ES) m/z 510(M+H)

Synthesis of N-trityl deoxynucleosides5′,3′-O-(Tetraisopropyldisiloxane-1,3-diyl) deoxynucleoside (10 mmole)was coevaporated 3 times with pyridine, and then dried on vacuum pumpfor 12 hours. Anhydrous pyridine (30 mL) and trityl chloride (11 mmole)were added, and the mixture was stirred at room temperature until TLC(CHCl₃/MeOH 9:1) showed full disappearance of nucleoside substrate(16-24 hours). The reaction was quenched with water/ice. Crude productwas extracted with DCM, washed with 5% aqueous solution of NaHCO₃, anddried with anhydrous Na₂SO₄. After filtration the organic layer wasconcentrated to dryness and left on vacuum pump for 3 hours. Hydrogenfluoride_(aq) (1.4 mL, 35 mmole) was carefully added (with vigorousstirring) to ice-cold solution of TEMED (7.5 mL, 50 mmole) inacetonitrile (20 mL). The TEMED-HF reagent so formed was thentransferred via teflon tubing to the flask with5′,3′-O-(tetraisopropyldisiloxane-1,3-diyl) N-trityl protecteddeoxynucleoside (10 mmole), and the mixture was stirred for 2.5 hours.The solution was concentrated, the residue coevaporated with each:pyridine, toluene, and EtOH. The crude product was purified by silicagel column chromatography using CHCl₃/Py (99.9:0.1) with a gradient ofmethanol (0-8%).

-   B=Cyt^(DMT): yield 98%; MS (ES) m/z 530(M+H), 552(M+Na), 568(M+K)-   B=Cyt^(TMT): yield 98%; MS (ES) m/z 560(M+H), 566(M+Li), 582(M+Na),    598(M+K), 692(M+Cs)-   B=Ade^(DMT): yield 91%; MS (ES) m/z 554(M+H)-   B=Ade^(MMT): yield 99.9%; MS (ES) m/z 524(M+H)-   B=Gua^(DMT): yield 70.4%; MS (ES) m/z 570(M+H)

5′-Carbonate protection of N-trityl deoxynucleosides

Synthesis of 5′-O-[3-(trifluoromethyl)phenoxy]carbonyl N-trityldeoxynucleosides

N-Trityl protected deoxynucleoside (5 mmole) was coevaporated 3 timeswith pyridine, dried on vacuum pump for 16 hours, and then dissolved inanhydrous pyridine (50 mL). The solution was cooled in dry ice/EtOHbath, and 3-(trifluoromethyl)phenyl chloroformate (1.19 g, 5.25 mmole)was added. The cooling bath was removed, the reaction mixture was shakenuntil all the reactant was completely dissolved, and then left overnightwith stirring. The reaction was quenched with water. The product wasextracted with DCM, washed with 5% aqueous solution of NaHCO₃, and driedwith anhydrous Na₂SO₄. The product was purified by silica gel columnchromatography with CHCl₃/benzene (9:1), followed by slow gradient from1% to 5% of methanol in CHCl₃ containing 0.1% Py.

-   B=Cyt^(DMT): yield 73%; HRMS (FAB) calc'd for C₃₈H₃₄N₃O₈F₃ (M+)    717.2298, found 717.2321-   B=Cyt^(TMT): yield 46%; HRMS (FAB) calc'd for C₃₉H₃₆N₃O₉F₃ (M+)    747.2404, found 747.2378-   B=Ade^(DMT): yield 61%; HRMS (FAB) calc'd for C₃₉H₃₃N₅O₇F₃ (M−H)    740.2332, found 740.2331-   B=Ade^(MMT): yield 75%; MS (ES) m/z 712(M+H), 734 (M+Na), 750 (M+K)-   B=Gua^(DMT): yield 60%; HRMS (FAB) calc'd for C₃₉H₃₅N₅O₈F₃ (M+H)    758.2438, found 758.2473

Phosphitylation of 5′-carbonate protected deoxynucleosides

Synthesis of deoxynucleoside3′-O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidites

The protected deoxynucleoside (3 mmole) and tetrazole (210 mg, 3 mmole)were dried separately on a vacuum pump for 16 hours. Deoxynucleoside wasdissolved in anhydrous DCM (30 mL), and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphane (950 mg, 3.15 mmole) wasadded in one portion. Tetrazole was added slowly to the reaction mixtureover 1 hour. The reaction mixture was then stirred for another 3 hours.A small amount of Et₃N (approx. 0.5 mL) was added to neutralize thesolution. The solvent was removed in vacuo, and the crude productchromatographed with benzene followed by a gradient of EtOAc (0-40 or60%) in benzene containing 0.1% Et₃N.

-   B=Cyt^(DMT): yield 61%; ³¹P NMR (CDCl₃) δ 150.21, 150.10; HRMS (FAB)    calc'd for C₄₇H₅₁N₅O₉F₃ (M+) 917.3377, found 917.3346-   B=Cyt^(TMT): yield 71%; ³¹P NMR (CDCl₃) δ 150.18, 150.09; HRMS (FAB)    calc'd for C₄₈H₅₃N₅O₁₀F₃ (M+) 947.3482, found 947.3503-   B=Ade^(DMT): yield 74%; ³¹P NMR (CDCl₃) δ 150.13, 150.05; HRMS (FAB)    calc'd for C₄₇H₅₁N₇O₈F₃ (M+) 941.3489, found 941.3510-   B=Ade^(MMT): yield 72%; MS (ES) m/z 912(M+H), 934 (M+Na), 950 (M+K)-   B=Gua^(DMT): yield 48%; ³¹P NMR (CDCl₃) δ 150.00, 149.88; HRMS (FAB)    calc'd for C₄₈H₅₂N₇O₉F₃ (M+H) 958.3516, found 958.3556

It will be apparent to one of skill in the art that the series ofsyntheses described above may be altered to employ analogous startingmaterials that react in a similar manner to give analogous products, andthat such alteration of the synthesis is within ordinary skill in theart. For example, the base “B” in the synthesis may be selected fromnucleobases, modified nucleobases, other heterocyclic bases, andanalogues thereof, provided the base “B” has an exocyclic amine group.As another example, the protecting group may be located on the3′-hydroxyl and the reactive phosphorus moiety may then be located onthe 5′-hydroxyl. As further example, the 5′-hydroxylprotecting group maybe replaced with a different protecting group, such asphenyloxycarbonyloxy, p-(phenylazo)phenyloxycarbonyl-oxy,o-nitrophenyloxycarbonyloxy, 9-fluorenylmethoxycarbonyloxy,2,2,2-Trichloro-1,1-dimethylethoxycarbonyloxy,4-chlorophenyloxycarbonyloxy,bis(trimethylsiloxy)-cyclobenzhydroxysilyloxy,bis(trimethylsiloxy)cyclododecyloxysilyloxy, and the like.

Synthesis on the Solid Support

The deoxynucleoside3′-O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidites synthesizedabove were used for the synthesis of polynucleotides. Synthesis ofpolynucleotides was conducted on a solid support as described hereinwith the following specified conditions:

-   -   Monomer concentration is 0.1 Molar solution in MeCN    -   Activator: Tetrazole (0.45 Molar solution in MeCN)    -   Cycle: 300 sec coupling        -   2.5, 5 or 7 min deprotection (removal of the            5′-hydroxylprotecting group) using a buffered aqueous            solution of peroxyanions

-   1. Trityl deprotection (3% TCA/DCM, 15 min to 2 hours, RT)

-   2. Cleavage from the support (conc.NH₄OH, 2 hours, RT)

-   RP-HPLC analysis:

-   ODS-Hypersil (5μ) column, flow 1.5 mL/min

-   0-20% MeCN in 50 mM TEAB (linear gradient) in 40 min

Products synthesized according to the method of the present inventionincluded: (AT)₅ (SEQ ID NO:1); A₉T (SEQ ID NO:2); (CT)₅ (SEQ ID NO:3);and C₉T (SEQ ID NO:4). Reverse-phase HPLC analysis demonstrated anacceptable product that typically was equivalent or superior to similarproducts synthesized using the conventional 4-step process.

The triaryl methyl protecting groups can be individually modified tooptimize their use for each of the naturally occurring nucleobases orfor use with modified nucleobases. The use of triaryl methyl protectinggroups that are resistant to strong nucleophiles can prevent or reducecertain undesirable types of nucleobase modifications such asde-amination (loss of the amine group) and depurination (loss of thepurine base). In addition, the triaryl methyl protecting group typicallyimparts a great deal of hydrophobicity to the precursor. Thishydrophobicity aids in the solubility of the precursor in anhydroussolvents required for effective coupling.

While the foregoing embodiments of the invention have been set forth inconsiderable detail for the purpose of making a complete disclosure ofthe invention, it will be apparent to those of skill in the art thatnumerous changes may be made in such details without departing from thespirit and the principles of the invention. Accordingly, the inventionshould be limited only by the following claims.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties, provided that, ifthere is any conflict in definitions, the present specification shall becontrolling.

This invention was made with Government support under Agreement No.N39998-01-9-7068. The Government has certain rights in the invention.

1. A method of forming an internucleotide bond, the method comprising: contacting a functionalized support with a precursor under conditions and for a time sufficient to result in internucleotide bond formation, said functionalized support comprising a solid support, a triarylmethyl linker group, and a nucleoside moiety having a reactive site hydroxyl, the nucleoside moiety bound to the solid support via the triarylmethyl linker group, said precursor having the structure (IVc)

wherein: O and H represent oxygen and hydrogen, respectively; R₁ is hydrido, hydroxyl, protected hydroxyl, lower alkyl, substituted lower alkyl, or alkoxy, one of R₂ or R₃ is a hydroxyl protecting group; and the other of R₂ or R₃ is a reactive group capable of reacting with a reactive site hydroxyl of a nucleoside moiety to produce either a H-phosphonite diester or a phosphite triester internucleotide linkage; Base is a heterocyclic base having an exocyclic amine group; and Tram is an abbreviation representing an exocyclic amine group protected by a triarylmethyl protecting group; wherein said functionalized support has the structure (Ic)

wherein:

is the solid support, Lnk′—is a linking group linking the solid support and the triaryl methyl linker group, or is a bond linking the solid support and the triaryl methyl linker group Trl—is the triarylmethyl linker group having three aryl groups, wherein each of the three aryl groups bound to a central methyl carbon, and at least one of said three aryl groups has one or more substituents, wherein one of said substituents is bound to the solid support and the central methyl carbon is bound to the 5′-hydroxyl of a nucleosidyl moiety either directly or through Lnk, (Ntd)_(k)—is a polynucleotide moiety having k nucleotide sub-units, wherein k is an integer in the range from zero to about 200, Lnk—is a linking group linking the triarylmethyl linker group and the polynucleotide moiety, or is a bond linking the triarylmethyl linker group and the polynucleotide moiety, and Nucl—is a nucleoside moiety having an unprotected 3′ or 5′ hydroxyl.
 2. The method of claim 1, wherein the exocyclic amine triaryl methyl protecting group has the structure (VIc)

wherein the broken line represents a C—N bond between the amino nitrogen of the exocyclic amine-attached group and the central methyl carbon of said triaryl methyl protecting group, and R₄, R₅ and R₆ are independently selected from unsubstituted and substituted aryl groups.
 3. The method of claim 2, wherein R₄, R₅, and R₆ are independently selected from substituted and unsubstituted phenyl groups.
 4. The method of claim 2, wherein R₄, R₅, and R₆ are optionally substituted aryl groups independently selected from phenyl, biphenyl, naphthanyl, indolyl, pyriduiyl, pyrrolyl, thiophenyl, furanyl, annulenyl, quinolinyl, and anthracenyl.
 5. The method of claim 2, wherein R₄, R₅, and R₆ are independently selected from phenyl, methoxyphenyl, dimethoxyphenyl, trimethoxyphenyl, and furanyl.
 6. The method of claim 1, wherein Ris a hydrido, the hydroxyl protecting group is a carbonate protecting group, and the reactive group has the structure (Vc):

wherein: the broken line indicates the bond to the sugar group; X is selected from a halo group and a secondary amino group; and Y is selected from hydrido, hydrocarbyl and substituted hydrocarbyl.
 7. The method of claim 6, wherein X is a secondary amino group having the structure —NQ1Q2; in which Q1 and Q2 are independently selected from the group consisting of alkyl, aryl, aralkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, and cycloalkynyl.
 8. The method of claim 6, wherein Y is selected from alkyl, lower alkyl, alkenyl, benzyl, substituted benzyl, aryl, aralkyl, cycloalkyl, electron-β-(electron-withdrawing group) substituted alkyl, electron-β-(electron-withdrawing group) substituted ethyl; electron-withdrawing substituted phenyl; or electron-withdrawing substituted phenylethyl.
 9. The method of claim 6, wherein X is a diisopropyl amino group and Y is a cyanoethyl group.
 10. The method of claim 6, wherein X is a diisopropyl amino group and Y is selected from methyl, benzyl, substituted benzyl, β-cyanoethyl, methyl-β-cyanoethyl, dimethyl-β-cyanoethyl, phenylsulfonylethyl, methyl-sulfonylethyl, p-nitrophenylsulfonylethyl, 2,2,2-trichloro-1,1-dimethylethyl, 2-(4-pyridyl)ethyl, 2-(2-pyridyl)ethyl, allyl, 4-methylene-1-acetylphenol, β-thiobenzoylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl, 2,2,2-trichloroethyl, p-nitrophenylethyl, p-cyanophenyl-ethyl, 9-fluorenylmethyl, 1,3-dithionyl-2-methyl, 2-(trimethylsilyl)ethyl, 2-methylthioethyl, 2-(diphenylphosphino)-ethyl, 1-methyl-1-phenylethyl, 3-buten-1-yl, 4-(trimethylsilyl)-2-buten-1-yl, cinnamyl, αmethylcinnamyl, and 8-quinolyl.
 11. The method of claim 10, wherein the carbonate protecting group is an optionally substituted aryl carbonate protecting group. 